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Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica
Accepted Manuscript Title: Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica Author: Jamaluddin Ahamad Sandeep Ojha Ankita Srivastava Alok Bhattacharya Sudha Bhattacharya PII: DOI: Reference:
S0166-6851(15)30017-7 http://dx.doi.org/doi:10.1016/j.molbiopara.2015.07.006 MOLBIO 10915
To appear in:
Molecular & Biochemical Parasitology
Received date: Revised date: Accepted date:
10-6-2015 29-7-2015 31-7-2015
Please cite this article as: Ahamad Jamaluddin, Ojha Sandeep, Srivastava Ankita, Bhattacharya Alok, Bhattacharya Sudha.Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica.Molecular and Biochemical Parasitology http://dx.doi.org/10.1016/j.molbiopara.2015.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Post-transcriptional regulation of ribosomal protein genes
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during serum starvation in Entamoeba histolytica
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Jamaluddin Ahamad 1, Sandeep Ojha1, Ankita Srivastava1, Alok Bhattacharya2 and Sudha
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Bhattacharya1*
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1
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India
School of Environmental Sciences, Jawaharlal
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2
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Jamaluddin Ahamad ([email protected])
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Sandeep Ojha ([email protected])
School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
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Ankita Srivastava ([email protected])
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Alok Bhattacharya ([email protected])
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Sudha Bhattacharya ([email protected])
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* Corresponding author
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Prof. Sudha Bhattacharya
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School of Environmental Sciences,
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Jawaharlal Nehru University,
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New Delhi-110067, India
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E-mail: [email protected]; [email protected]
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Telephone: 91-11-26704308
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Nehru University, New Delhi-110067,
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Graphical abstract
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Graphical Abstract
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In E. histolytica, the mRNAs of ribosomal protein genes accumulate but translation is
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downregulated during serum starvation. Mutations upstream of AUG in the RPL30 gene relieve
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repression, suggesting post-transcriptional regulation.
29 Post-transcriptional regulation of RPL30 in E. histolytica RPL30 Serum starvation Transcripts persist
Translation declines 5’ UTR
serum starved N
4
8
16 24 hr
Mut
WT AUG
//
-9 TGCCAAAG -2
Luciferase activity/µg of protein x 10000
Translation repressed
33 34 35 36
//
-9 CATTGGGA -2
Translation repression relieved
35 30 25 20 15 10 5 0
WT Mut
N
30 31 32
AUG
4 hr 8 hr 16 hr 24 hr Serum starvation
37 38 39 40 41 42 43 44
Highlights
In E. histolytica, unprocessed pre-rRNA and RP mRNAs accumulate during serum starvation. Translation of RPS19 and RPL30 genes is downregulated during serum starvation suggesting post-transcriptional regulation. Mutations in the sequence -2 to -9 upstream of AUG in the RPL30 gene relieved translation repression during stress. E. histolytica stores untranslated RP mRNAs during stress.
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Abstract
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Ribosome synthesis involves all three RNA polymerases which are co-ordinately regulated to
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produce equimolar amounts of rRNAs and ribosomal proteins (RPs). Unlike model organisms
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where transcription of rRNA and RP genes slows down during stress, in E. histolytica rDNA
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transcription continues but pre-rRNA processing slows down and unprocessed pre-rRNA
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accumulates during serum starvation. To investigate the regulation of RP genes under stress we
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measured transcription of six selected RP genes from the small- and large ribosomal subunits
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(RPS6, RPS3, RPS19, RPL5, RPL26, RPL30) representing the early-, mid-, and late stages of
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ribosomal assembly. Transcripts of these genes persisted in growth-stressed cells. Expression of
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luciferase reporter under the control of two RP genes (RPS19 and RPL30) was studied during
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serum starvation and upon serum replenishment. Although luciferase transcript levels remained
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unchanged during starvation, luciferase activity steadily declined to 7.8% and 15% of control
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cells, respectively. After serum replenishment the activity increased to normal cells, suggesting
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post-transcriptional regulation of these genes. Mutations in the sequence -2 to -9 upstream of
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AUG in the RPL30 gene resulted in the phenotype expected of post-transcriptional regulation.
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Transcription of luciferase reporter was unaffected in this mutant, and luciferase activity did not
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decline during serum starvation, showing that this sequence is required to repress translation of
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RPL30 mRNA, and mutations in this region relieve repression. Our data show that during serum
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starvation E. histolytica blocks ribosome biogenesis post-transcriptionally by inhibiting pre-
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rRNA processing on the one hand, and the translation of RP mRNAs on the other.
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Keywords: Entamoeba histolytica; Pre-rRNA accumulation; Ribosomal proteins; Post-
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transcriptional regulation; 5’ Untranslated region (5’ UTR)
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1. Introduction
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Ribosome biogenesis in any cell type is a highly energy consuming process. It requires the
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coordinated regulation of all three RNA polymerases (Pol І, ІІ, and ІІІ) to produce equimolar
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amounts of the four rRNAs (18S, 5.8S, 28S and 5S rRNAs), ~80 ribosomal proteins (RPs) and
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more than 200 additional proteins [1, 2]. In most model organisms rDNA transcription ceases
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under conditions of growth stress, as the requirement for ribosomes goes down [3, 4]. We find
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that this is not the case in the early branching protest Entamoeba. We have earlier shown that in
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Entamoeba histolytica, a human parasite, rRNA transcription continues during serum starvation
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but the processing of pre-rRNA slows down, and unprocessed pre-rRNA accumulates to high
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levels [5]. In Entamoeba invadens, a reptilian parasite, when trophozoites are transferred to low
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glucose medium to induce cyst formation, unprocessed pre-rRNAs accumulate to high levels in
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the cyst, along with the RP gene transcripts [6, 7]. Thus Entamoeba has evolved somewhat
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different mechanisms to regulate ribosome biogenesis during cellular growth stress and
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differentiation.
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In model organisms there is crosstalk between the transcription machinery of RP and rRNA
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genes so that both may be coordinately regulated during growth stress [8, 9]. Studies with
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Saccharomyces cervisiae have demonstrated a number of mechanisms leading to coordinate
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regulation of RP and rRNA genes [10]. One of them is mediated by the transcription factors-
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Rap1, Fhl1, and Ifh1 needed for RP gene transcription. Ifh1 is part of a complex with CK2,
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Utp22 and Rrp7 (CURI). Of these Utp22 and Rrp7 are essential for pre-rRNA processing.
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During unfavourable growth conditions when rRNA synthesis slows down, free Utp22 and Rrp7
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sequester CK2 and Ifh1 in the CURI complex, and non-availability of Ifh1 slows down RP gene
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transcription. Conversely, depletion of Utp22 and Rrp7 results in increased levels of RP mRNA
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[11]. Another possible mechanism could be through the high mobility group (HMG) protein,
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Hmo1 (high mobility group protein1), which binds both to promoters of RP genes and to the
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rRNA gene locus [12]. The target of rapamycin (TOR) pathway which controls cell growth and
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proliferation in response to environmental signals could also contribute to the cross talk. In S.
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cerevisiae it has been shown that this pathway regulates H3K56 (histone H3 lysine56)
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acetylation which, in turn, regulates the binding of Hmo1 to rDNA [13].
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Coordinate regulation of rRNA and RP gene expression has not been studied in parasites. Since
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unprocessed pre-rRNA accumulates in E. histolytica during serum starvation we were interested
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to know whether RP mRNAs also accumulate under the same conditions, and whether these
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mRNAs are translated. Here we show that RP mRNAs persist during starvation but their
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translation is inhibited by sequences in the 5’ untranslated region (UTR).
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2. Materials and methods
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2.1. Cell culture and growth conditions
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Trophozoites of E. histolytica strain HM-1: IMSS (clone 6) were axenically maintained in TYI-
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S-33 medium supplemented with 15% adult bovine serum (PAA laboratories, Austria),
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Diamond’s Vitamin Tween 80 solution (Sigma-Aldrich) and antibiotics (0.3 units/ml penicillin
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and 0.25mg/ml streptomycin) at 35.5°C. For serum starvation, media from mid log phase grown
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trophozoites were replaced with TYI-S-33 medium containing 0.5% adult bovine serum for
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indicated time period. Replenishment was achieved by decanting total media after indicated time
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period and filled with complete TYI-S-33 medium for indicated time periods. G-418 (Sigma)
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was added at 10 µg/ml for maintaining the transfected cell lines [14].
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2 . 2 . RN A is o la t i o n a n d n o rt h e rn h y b r i d i z a t i o n
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E. histolytica trophozoites were grown for 48 hr and transferred to low-serum medium for serum
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starvation. Cells were removed at different time points. Total RNA from ∼5×106 cells was
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purified using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Poly
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A+RNA was isolated using poly A Tract mRNA isolation system (Promega) as per
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manufacturer’s protocol. For northern analysis 10-15 µg of total RNA was resolved on 1.2%
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formaldehyde agarose gel in gel running buffer [0.1 M MOPS (pH 7.0), 40 mM sodium acetate,
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5 mM EDTA (pH 8.0)] and 37% formaldehyde at 4 V/cm. The RNA was transferred on to
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GeneScreen plus R membrane (Perkin Elmer). α- P32dATP labeled probe was prepared by
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random priming method using NEBlot kit (NEB). Hybridization and washing conditions for
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RNA blots were as per manufacturer instructions.
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2 . 3 . L u c i f e ra s e r e p o rt e r c o n s t ru c t a n d s t a b le t r a n s f e c t i o n
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Upstream sequence (900bp) of RPS19 and RPL30 genes were amplified from genomic DNA of
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E. histolytica by PCR using primers AJF and AJR2, and ALF and ALR1 (all primers used in this
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study are listed in Supplementary Table 1). Primers ALF4 and ALR1 were used for amplifying
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the wild type (244bp) upstream sequence of RPL30 gene for luciferase expression. To mutate the
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motif1 (Mut 1) an amplicon of 219bp was obtained with primers ALF4 and ALR2M and an
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amplicon of 151bp was obtained with primers ALF5 and ALR which overlap by 27bp. (Primers
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ALR2M and ALF5 contained the mutated sequence). The two amplicons were stitched and a
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344bp fragment was obtained with ALF4 and ALR primers. Finally, using this fragment as a
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template, a 244bp fragment was amplified by using primers ALF4 and ALR1. A schematic
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description is given in supplementary Fig. S3. To mutate the motif 3 (Mut 2) of RPL30, a 244bp
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fragment was amplified by using primers ALF4 and ALR3 ACC (latter contained the mutated
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motif 3). To mutate the 5’UTR motif (Mut 3), a 244 bp fragment was amplified by using primers
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ALF4 and ALR MUT (latter contained mutated 5’UTR). Constructs were cloned upstream of
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LUC gene at XhoI/Acc651 site in pEh-NEO-LUC vector. Constructs were transfected by
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electroporation and maintained in presence of G418 at 10µg/ml [14].
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2.4. Luciferase assay
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This was done as described previously [15]. Briefly, stably transfected trophozoites, maintained
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in TYI-S-33 medium supplemented with 10 μg/ml G-418, were chilled on ice, harvested and
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washed twice in 1XPBS (pH 7.4), and lysed in 200 μl of reporter lysis buffer (Promega) with the
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addition of protease inhibitors E64-C and leupeptin. Lysates were frozen overnight at −80 °C.
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After thawing on ice for 10 min, cellular debris was pelleted, and the samples were allowed to
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warm to room temperature. Luciferase activity was measured according to the manufacturer's
151
instructions (Promega) using a Turner Luminometer (model TD-20E). Luciferase activity per
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microgram of protein was calculated as a measure of reporter gene expression.
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2.5. Prime r e xtension
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DNase I (Roche)-treated PolyA+ RNA (2µg) was used for primer extension with end labeled
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oligonucleotides (listed in Supplementary Table 1). Reverse transcription was carried out using
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superscript III (Invitrogen) at 45°C according to manufacturer's instructions. Sequencing reaction
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was run on 6% denaturing polyacrylamide gel. The gel image was produced using a Typhoon
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phosphor imager (GE Healthcare).
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2.6. Search for the motifs in upstream region of RP genes
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The online tool MEME [16] was used for prediction of the motifs present in upstream sequence.
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100 bp of sequence upstream of the RP’s were extracted from E. histolytica HM-I:IMSS genome
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and were analysed for the motifs. The input consisted of 188 FASTA formatted sequences of
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RPs with the default settings of width (minimum six and maximum 20) and the search was
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optimized for identifying zero or one motif for sequence and motif searched for given strand
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only. For control we used 150 bp upstream sequence to CDS of all E. histolytica HM-I:IMSS
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8333 genes using Perl- coding. The motifs were searched in these sequences using CisFinder
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with default parameters except the search was done for forward strand only and maximum
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number of motif to find were set as 100 (minimum for CisFinder).
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3. Results
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3.1. Ribosomal protein transcripts persist during serum starvation.
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Previous work had shown that unprocessed pre-rRNA accumulates in E. histolytica cells
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subjected to growth stress due to serum starvation [5]. We were interested to investigate whether
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transcripts of RP genes also persist during serum starvation in E. histolytica.
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The small (40S) and large (60S) ribosomal subunits are individually made by precise assembly
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of RPs and rRNAs. The synthesis and turnover of RPs is highly controlled to generate equimolar
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amount of all RPs [19, 20]. We selected three genes each from the small and large ribosomal
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subunits (RPS6, RPS3, RPS19, and RPL5, RPL26, RPL30) based on the stage (early, mid or
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late) at which the protein is thought to be assembled in the newly-forming ribosome [21].
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Transcript levels were measured in normal and serum-starved cells, by northern hybridization
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with total RNA (Fig. 1). Since each of these genes is present in multiple copies in E. histolytica,
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the accession number of the copy selected for this study has been indicated in Fig. 1. Multiple
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copies of the same gene have 97 - 100% amino acid sequence identity and >92% nucleotide
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sequence identity. Each gene probe hybridized with a single band of size expected from the
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predicted ORF, since UTRs in E. histolytica are generally very short [22]. The transcript levels
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of none of the six RP genes changed significantly during serum starvation. These data suggest
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that like pre-rRNA, the transcripts of RP genes also persist at normal levels during serum
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starvation.
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3.2. Translation of luciferase reporter (driven by RP genes) is down regulated during
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serum starvation.
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Genes encoding the components of translational apparatus are often the targets of post-
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transcriptional regulation [23]. Since mRNA levels of RP genes did not decline during serum
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starvation, we were interested to investigate whether translation of these mRNAs continues
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during serum starvation. For this, we cloned 900bp of the 5’-upstream sequence (upstream from
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the first AUG codon) of two of the six RP genes studied above (RPS19 and RPL30) to drive a
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luciferase reporter gene, using Eh-Neo-Luc vector. (The 900 bp upstream sequence included, in
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addition to the intergenic region, the upstream gene sequence at each locus as well). Stable
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transfectants were obtained by G418 selection and the levels of luciferase transcripts were
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measured by northern hybridization in cells undergoing serum starvation. As observed for the
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RPS19 and RPL30 transcripts, luciferase transcripts driven from these promoters also persisted
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in starved cells and there was no significant change in transcript levels (Fig. 2 a, b). Further,
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transcript levels were measured after serum replenishment and, again, no significant change was
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observed (Fig. 2 c, d). To check whether mRNA translation might be affected during serum
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starvation, luciferase enzyme levels were measured in cells after shifting to low-serum medium.
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The data showed that in both cell lines the luciferase activity dropped steadily during serum
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starvation. After 24 hr of serum starvation the activity was only 7.8% of control in the RPS19
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cell line and 15% of control in the RPL30 cell line (Fig. 2 e, f). To show that this drop in activity
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was not due to general instability of luciferase in serum starved cells, we expressed the luciferase
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gene from the amebic lectin promoter and measured activity during serum starvation. Luciferase
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activity remained unchanged in these cells (Supplementary Fig. S1). From this we can conclude
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that translation of reporter luciferase transcript driven by RPS19 and RPL30 upstream sequences
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is down regulated during serum starvation. We further measured luciferase activity in starved
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cells after serum replenishment. The data showed that in both cell lines the luciferase activity
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started going up immediately on serum replenishment and reached almost normal levels by 6 hr
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(Fig. 2 g, h). Since the luciferase transcript levels in these cells had remained unchanged (Fig. 2
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c, d), this again confirms that luciferase expression driven by RPS19 and RPL30 upstream
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sequences is regulated post-transcriptionally.
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3.3. Analysis of conserved regulatory motifs in 5’ upstream sequences
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Translational regulation of mRNAs is most frequently mediated by sequences in the 5’UTR. We
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wished to identify conserved sequence motifs in the upstream sequences (upstream of the
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initiating AUG codon) of all E. histolytica RP genes, and test whether any of these may be
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involved specifically in down regulation of RP mRNA translation, without affecting transcription
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of the gene. We took 100bp of upstream sequences from all RP genes and searched for
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conserved motifs using MEME motif finder tool. Three conserved motifs were predicted
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(Supplementary Table 2). Motif 1was found in 75% of RP genes; motif 2 in 50% of RP genes;
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and motif 3in 69% of RP genes. These motifs were searched in upstream sequences of all E.
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histolytica genes. Motif 1 was present in 25%, motif 2 in 33%, and motif 3 in 53% of all genes.
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Thus, only motif 1 was significantly enriched in RP genes.
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We selected the RPL30 gene to study the effect of mutations in these motifs on transcription and
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translation. The luciferase reporter system was used for the purpose. Of the three conserved
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motifs the RPL30 gene contained motif 1 (nt position -52 to -58), and motif 3 (-24 to -29) (Fig. 3
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a). Motif 2 was not found. The transcription start site (TSS) of this gene was mapped by primer
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extension using a primer from position +79 to +99 with respect to AUG. The TSS was located at
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nt position -29, with respect to AUG (Supplementary Fig. S2). This coincided with the 5’ end of
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motif 3. Mutations were generated as follows (Supplementary Fig. S3). Part of motif 1,
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GAACCC, was mutated to AGGTTT (Mut 1); and motif 3, GAACTT, was mutated to AGGTCC
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(Mut 2). Constructs were generated with luciferase gene cloned downstream of the two mutated
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sequences, and luciferase expression was measured in stable transfectants grown under normal
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and serum-starved conditions. Mut 1 resulted in a severe transcription defect and the level of
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luciferase transcript dropped to ~15% of WT under normal growth conditions, with further
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decrease during serum starvation (Fig. 3 c, f). Transcription of luciferase reporter was less
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affected in Mut 2 than Mut 1, but was only~45% compared with WT. These transcripts persisted
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during serum starvation, with slight decrease at 24 hr as seen in WT cells (Fig. 3 d, f). Luciferase
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activity was very low in Mut 1 cells, as expected, and in Mut 2 cells it was ~25% of WT under
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normal growth conditions (Fig. 3 g). When cells were subjected to serum starvation, luciferase
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activity steadily declined in WT cells. In Mut 2 cells the activity declined initially, but went up to
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the level in unstarved Mut 2 cells (Fig. 3 g). Since this activity was comparable to that in 24 hr-
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starved WT cells, it is not possible to comment whether motif 3 may have a role in translational
248
down regulation during starvation. However mutations in this motif did have a negative impact
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on transcription and/or stability of the transcript.
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3.4. The sequence immediately upstream of AUG (-2 to -9) is involved in translational down
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regulation during stress
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To understand the mechanism by which translation of RP genes is controlled during stress, one
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would need mutants which are specifically affected in translation but show normal transcription,
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which was not the case in the mutants described above. It has been reported in Arabidopsis and
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yeast that sequences immediately upstream of AUG are important modulators of translation
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efficiency [24, 25]. To test whether the same may be true in the RP genes of E. histolytica, we
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generated a mutation (Mut 3) at position -2 to -9 just upstream of the AUG in RPL30 gene where
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the WT sequence TGCCAAAG was changed to CATTGGGA. (The -2 to -9 WT sequence is
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identical in both copies of the RPL30 gene). The mutation was generated as described in
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Supplementary Fig. S3. Unlike previous constructs in which 900 bp of upstream sequence had
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been used, the Mut 3 construct contained 244 bp of upstream sequence which covered the entire
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intergenic sequence of 242 bp, but excluded the upstream gene. This mutant had normal levels of
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luciferase transcripts in unstarved cells (Fig. 3 e, f), and the transcripts persisted at the same
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levels till 16 hr of serum starvation, after which there was a slight decline in both mutant and WT
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cells. The luciferase activity in these cells was comparable to WT in unstarved cells, but unlike
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WT cells it remained high even after 24 hr of serum starvation (Fig. 3 g). Thus the sequence -2 to
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-9 in the 5’ UTR of the RPL30 gene is required to repress translation of RPL30 mRNA in serum
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starved cells, and mutations in this region relieve repression.
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Taken together our data show that during serum starvation E. histolytica blocks ribosome
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biogenesis post-transcriptionally by inhibiting pre-rRNA processing on the one hand, and the
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translation of RP mRNAs on the other. Translational inhibition of RPL30 mRNA is controlled
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by sequences immediately upstream of AUG.
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4. Discussion
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The transcription of rRNA and RP genes slows down or stops during growth stress in most
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model organisms [26, 3]. We have earlier shown that in E. histolytica unprocessed pre-rRNA, in
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fact, accumulates to high levels during serum starvation [5]. Here we show that transcripts of
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genes encoding both the small- and large-ribosomal subunit proteins also persist during serum
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starvation, suggesting that the early branching protist E. histolytica has adopted a post-
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transcriptional mechanism to regulate ribosome biogenesis whereby it stores pre-rRNA in an
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unprocessed state and RP mRNAs in an untranslated state during growth stress. Such a
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regulatory mechanism would provide the stressed E. histolytica trophozoite with a large stock of
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RNA intermediates, to be rapidly utilized for assembly of mature ribosomes when conditions are
283
favorable for growth. It is possible that reinitiation of transcription from a silenced template may
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be a slow process in Entamoeba and therefore gene copies are kept transcriptionally active even
285
when growth slows down.
286
Our data clearly show translational down regulation of RPS19 and RPL30 genes in starved cells.
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Each of these genes is present in two copies in E. histolytica. The copies of RPS19 show 98%
288
identity and those of RPL30 show 97% identity at amino acid level. The upstream sequences of
289
the two gene copies in each case have no similarity as they occupy different genomic locations.
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It is, therefore, possible that each copy is subject to differential translational regulation.
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However, our further analysis of RPL30 showed that mutations of upstream sequence motifs
292
adversely influenced transcription, while the 5’UTR sequence, which is identical in the two
293
RPL30 gene copies, was involved in translational regulation. The 5’UTR of mRNAs is typically
294
one of the most important determinants to modulate translation efficiency. Both in vertebrates
295
and yeast many of the translationally controlled genes have longer 5’UTRs which may form
296
stable stem-loop structures that impede scanning and resist unwinding by translation initiation
297
factors [27]. Sequence-specific binding of trans-acting factors to secondary structure elements in
298
the 5’UTR would inhibit translation [28], which has been well-documented for ferritin mRNA
299
[29]. It is possible that a trans-acting factor binds to the 5’UTR during serum starvation and
300
inhibits translation of the RPL30 gene of E. histolytica. If such an interaction is sequence-
301
specific one may expect to find conserved motifs in 5’UTR and upstream sequences of all RP
302
genes to enable their coordinate control. We found only one upstream motif (motif 1) that could
303
be considered specific to RP genes as it was present in most of them, and was infrequent in other
304
E. histolytica genes. However, at least in the case of RPL30 gene this motif was involved in
305
transcription, rather than translational control. On the other hand we found the involvement of a
306
sequence immediately upstream of AUG in translational control of the RPL30 gene. The
307
importance of nucleotides immediately upstream of AUG in translational regulation has been
308
revealed from studies with Arabidopsis [24] and yeast [25]. In Arabidopsis it was shown that
309
translational efficiency of genes varied over 200-fold depending on their 5’UTR sequences, and
310
the most critical positions were -5 to -1. A-residues at these positions favoured translation while
311
T-residues were repressive [24]. In yeast the positions -3 to -1 upstream of AUG were the most
312
important, and here too the favoured nt was A. Our data show that in E. histolytica the sequence
313
-9 to -2 upstream of AUG in the RPL30 gene was crucial in translational regulation during serum
314
starvation. Both the WT sequence (TGCCAAAG) which repressed translation, and the mutant
315
sequence (CATTGGGA) which relieved the repression, contained A residues at different
316
positions. The contribution of each of these nucleotide residues to translational repression needs
317
to be determined. A search of the -9 to -2 WT sequence (TGCCAAAG) of RPL30 did not reveal
318
identical motifs in 5’UTRs of other RP genes. It is possible that the regulatory sequence motif
319
may be very short, or that the secondary structure of the 5’UTR rather than its sequence may be
320
the important determinant.
321
A common mechanism of translational regulation of genes involved in ribosome biogenesis
322
exists in vertebrates. The mRNAs of all RP genes and other genes coding for components of the
323
translational apparatus contain a 5’terminal oligo pyrimidine (TOP) motif [30]. TOP mRNAs
324
typically initiate with a C-residue followed by an uninterrupted stretch of 4-14 pyrimidines.
325
These mRNAs are translationally repressed under unfavourable growth conditions, and exist as
326
mRNPs. Apart from vertebrates TOP mRNAs have been reported in Drosophila melanogaster,
327
but have not yet been reported in other model organisms like Caenorhabditis elegans and yeast
328
[30]. Thus it is not clear whether this elegant control mechanism is universal. Since the TSS of E.
329
histolytica RP genes has not been mapped it is not possible to determine whether the TOP motif
330
exists in this organism. Our primer extension mapping of RPL30 transcript showed the TSS to be
331
located 29 nt upstream at a G residue, which is incompatible with a TOP motif. We also looked
332
for poly pyrimidine stretches starting with C in the region -10 to -100 upstream of all RP genes.
333
This sequence should include the TSS of most genes as the 5’UTR of E. histolytica genes is
334
<100 nt [22].
335
Post-transcriptional control of gene expression has been reported in other human parasites. In
336
Plasmodium the CDPK1 gene translationally activates repressed mRNA species in the
337
developing zygote, and translation of these mRNAs is dependent on expression of CDPK1 [31].
338
Both the 5’ and 3’ UTRs are required for activation. Genes coding for proteins of the LCCL
339
complex in Plasmodium are transcribed but translationally repressed in gametocytes [32]. In
340
Leishmania the expression of Hsp83 gene is controlled post-transcriptionally through a poly
341
pyrimidine-rich element in the 3’UTR [33]. In Leishmania major, the RPL19 protein is
342
undetectable in stationary phase promastigotes and amastigotes, despite high transcript levels
343
[34]. Studies with the sequences flanking the E. histolytica RPL21 gene also showed that the
344
gene is regulated post-transcriptionally [35], although the sequences involved were not
345
identified. Our study documents the involvement of sequences immediately upstream of AUG in
346
translational regulation in E. histolytica. Such a mechanism has not been reported before, and it
347
will be interesting to know whether this may be a common regulatory mechanism controlling the
348
expression of ribosomal biogenesis genes in E. histolytica.
349
Competing interest
350
The authors declare that they have no competing interests.
351
Author’s contributions
352
SB proposed and designed the research, drafted the final version of the manuscript; AB designed
353
and analyzed the computational work. JA performed the computational and experimental work.
354
SO helped in Northern blotting. AS helped in computational analysis. All authors have
355
participated in preparing the manuscript. All authors have read and approved the final
356
manuscript.
357
Acknowledgements
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This work was supported by a grant from the department of Science and Technology, India to SB
359
and fellowship from University Grants Commission, India to JA, SO and AS.
360
References
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Fig. 1. Transcript levels of RP genes during serum starvation. (a & b) Total RNA was isolated at different time points during serum starvation and 15 µg RNA of each sample was electrophoresed (as described in Fig. 1a), blotted and hybridized with full length probes of indicated SSU and LSU RP genes (early, mid and late stage according to the time at which the protein gets assembled in the newly synthesized ribosome). The expected size of each transcript is indicated on the right. (c & d) The band intensity in northern blots was determined by densitometric scanning, and values were normalized with respect to loading control (18S rRNA) in each lane. The data is an average of three independent experiments. N, unstarved cells.
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Fig. 2. Levels of luciferase transcript and luciferase activity in RPS19 and RPL30 cell lines during serum starvation and replenishment. 5’-upstream sequences (900bp) of the RPS19 and RPL30 genes were amplified from genomic DNA of E. histolytica. Both the fragments were cloned at XhoI and Acc651 sites in pEhNeo-Luc vector. Stably transfected cells were grown for 48 hr and subjected to serum starvation for 24 hr, followed by serum replenishment. (a-d) Transcript levels were measured by northern hybridization with full length luciferase probe. (eh) Luciferase activity per µg of protein was measured by luciferase assay. Data presented is of three independent measurements.
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Fig. 3. Mutational analysis of conserved motifs in 5’upstream regulatory sequence of E. histolytica RPL30 gene. (a) The location of conserved motifs 1 and 3 in the RPL30 upstream sequence are indicated, along with their mutated sequences. The sequence of Mut 3 is marked. (b-e) Northern blot analysis of luciferase expressed from WT and mutant RPL30 sequences in serum starved (SS) cells. (f) Luciferase transcripts in unstarved mutant cell lines relative to WT, (‘N’ lanes in b-e) and (g) luciferase activity in WT and mutants during serum starvation.
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S0166-6851(15)30017-7 http://dx.doi.org/doi:10.1016/j.molbiopara.2015.07.006 MOLBIO 10915
To appear in:
Molecular & Biochemical Parasitology
Received date: Revised date: Accepted date:
10-6-2015 29-7-2015 31-7-2015
Please cite this article as: Ahamad Jamaluddin, Ojha Sandeep, Srivastava Ankita, Bhattacharya Alok, Bhattacharya Sudha.Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica.Molecular and Biochemical Parasitology http://dx.doi.org/10.1016/j.molbiopara.2015.07.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Post-transcriptional regulation of ribosomal protein genes
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during serum starvation in Entamoeba histolytica
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Jamaluddin Ahamad 1, Sandeep Ojha1, Ankita Srivastava1, Alok Bhattacharya2 and Sudha
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Bhattacharya1*
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1
6
India
School of Environmental Sciences, Jawaharlal
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2
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Jamaluddin Ahamad ([email protected])
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Sandeep Ojha ([email protected])
School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India
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Ankita Srivastava ([email protected])
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Alok Bhattacharya ([email protected])
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Sudha Bhattacharya ([email protected])
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* Corresponding author
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Prof. Sudha Bhattacharya
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School of Environmental Sciences,
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Jawaharlal Nehru University,
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New Delhi-110067, India
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E-mail: [email protected]; [email protected]
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Telephone: 91-11-26704308
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Nehru University, New Delhi-110067,
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Graphical abstract
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Graphical Abstract
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In E. histolytica, the mRNAs of ribosomal protein genes accumulate but translation is
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downregulated during serum starvation. Mutations upstream of AUG in the RPL30 gene relieve
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repression, suggesting post-transcriptional regulation.
29 Post-transcriptional regulation of RPL30 in E. histolytica RPL30 Serum starvation Transcripts persist
Translation declines 5’ UTR
serum starved N
4
8
16 24 hr
Mut
WT AUG
//
-9 TGCCAAAG -2
Luciferase activity/µg of protein x 10000
Translation repressed
33 34 35 36
//
-9 CATTGGGA -2
Translation repression relieved
35 30 25 20 15 10 5 0
WT Mut
N
30 31 32
AUG
4 hr 8 hr 16 hr 24 hr Serum starvation
37 38 39 40 41 42 43 44
Highlights
In E. histolytica, unprocessed pre-rRNA and RP mRNAs accumulate during serum starvation. Translation of RPS19 and RPL30 genes is downregulated during serum starvation suggesting post-transcriptional regulation. Mutations in the sequence -2 to -9 upstream of AUG in the RPL30 gene relieved translation repression during stress. E. histolytica stores untranslated RP mRNAs during stress.
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Abstract
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Ribosome synthesis involves all three RNA polymerases which are co-ordinately regulated to
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produce equimolar amounts of rRNAs and ribosomal proteins (RPs). Unlike model organisms
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where transcription of rRNA and RP genes slows down during stress, in E. histolytica rDNA
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transcription continues but pre-rRNA processing slows down and unprocessed pre-rRNA
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accumulates during serum starvation. To investigate the regulation of RP genes under stress we
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measured transcription of six selected RP genes from the small- and large ribosomal subunits
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(RPS6, RPS3, RPS19, RPL5, RPL26, RPL30) representing the early-, mid-, and late stages of
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ribosomal assembly. Transcripts of these genes persisted in growth-stressed cells. Expression of
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luciferase reporter under the control of two RP genes (RPS19 and RPL30) was studied during
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serum starvation and upon serum replenishment. Although luciferase transcript levels remained
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unchanged during starvation, luciferase activity steadily declined to 7.8% and 15% of control
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cells, respectively. After serum replenishment the activity increased to normal cells, suggesting
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post-transcriptional regulation of these genes. Mutations in the sequence -2 to -9 upstream of
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AUG in the RPL30 gene resulted in the phenotype expected of post-transcriptional regulation.
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Transcription of luciferase reporter was unaffected in this mutant, and luciferase activity did not
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decline during serum starvation, showing that this sequence is required to repress translation of
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RPL30 mRNA, and mutations in this region relieve repression. Our data show that during serum
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starvation E. histolytica blocks ribosome biogenesis post-transcriptionally by inhibiting pre-
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rRNA processing on the one hand, and the translation of RP mRNAs on the other.
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Keywords: Entamoeba histolytica; Pre-rRNA accumulation; Ribosomal proteins; Post-
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transcriptional regulation; 5’ Untranslated region (5’ UTR)
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1. Introduction
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Ribosome biogenesis in any cell type is a highly energy consuming process. It requires the
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coordinated regulation of all three RNA polymerases (Pol І, ІІ, and ІІІ) to produce equimolar
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amounts of the four rRNAs (18S, 5.8S, 28S and 5S rRNAs), ~80 ribosomal proteins (RPs) and
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more than 200 additional proteins [1, 2]. In most model organisms rDNA transcription ceases
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under conditions of growth stress, as the requirement for ribosomes goes down [3, 4]. We find
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that this is not the case in the early branching protest Entamoeba. We have earlier shown that in
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Entamoeba histolytica, a human parasite, rRNA transcription continues during serum starvation
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but the processing of pre-rRNA slows down, and unprocessed pre-rRNA accumulates to high
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levels [5]. In Entamoeba invadens, a reptilian parasite, when trophozoites are transferred to low
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glucose medium to induce cyst formation, unprocessed pre-rRNAs accumulate to high levels in
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the cyst, along with the RP gene transcripts [6, 7]. Thus Entamoeba has evolved somewhat
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different mechanisms to regulate ribosome biogenesis during cellular growth stress and
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differentiation.
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In model organisms there is crosstalk between the transcription machinery of RP and rRNA
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genes so that both may be coordinately regulated during growth stress [8, 9]. Studies with
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Saccharomyces cervisiae have demonstrated a number of mechanisms leading to coordinate
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regulation of RP and rRNA genes [10]. One of them is mediated by the transcription factors-
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Rap1, Fhl1, and Ifh1 needed for RP gene transcription. Ifh1 is part of a complex with CK2,
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Utp22 and Rrp7 (CURI). Of these Utp22 and Rrp7 are essential for pre-rRNA processing.
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During unfavourable growth conditions when rRNA synthesis slows down, free Utp22 and Rrp7
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sequester CK2 and Ifh1 in the CURI complex, and non-availability of Ifh1 slows down RP gene
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transcription. Conversely, depletion of Utp22 and Rrp7 results in increased levels of RP mRNA
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[11]. Another possible mechanism could be through the high mobility group (HMG) protein,
97
Hmo1 (high mobility group protein1), which binds both to promoters of RP genes and to the
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rRNA gene locus [12]. The target of rapamycin (TOR) pathway which controls cell growth and
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proliferation in response to environmental signals could also contribute to the cross talk. In S.
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cerevisiae it has been shown that this pathway regulates H3K56 (histone H3 lysine56)
101
acetylation which, in turn, regulates the binding of Hmo1 to rDNA [13].
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Coordinate regulation of rRNA and RP gene expression has not been studied in parasites. Since
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unprocessed pre-rRNA accumulates in E. histolytica during serum starvation we were interested
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to know whether RP mRNAs also accumulate under the same conditions, and whether these
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mRNAs are translated. Here we show that RP mRNAs persist during starvation but their
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translation is inhibited by sequences in the 5’ untranslated region (UTR).
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2. Materials and methods
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2.1. Cell culture and growth conditions
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Trophozoites of E. histolytica strain HM-1: IMSS (clone 6) were axenically maintained in TYI-
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S-33 medium supplemented with 15% adult bovine serum (PAA laboratories, Austria),
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Diamond’s Vitamin Tween 80 solution (Sigma-Aldrich) and antibiotics (0.3 units/ml penicillin
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and 0.25mg/ml streptomycin) at 35.5°C. For serum starvation, media from mid log phase grown
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trophozoites were replaced with TYI-S-33 medium containing 0.5% adult bovine serum for
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indicated time period. Replenishment was achieved by decanting total media after indicated time
115
period and filled with complete TYI-S-33 medium for indicated time periods. G-418 (Sigma)
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was added at 10 µg/ml for maintaining the transfected cell lines [14].
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2 . 2 . RN A is o la t i o n a n d n o rt h e rn h y b r i d i z a t i o n
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E. histolytica trophozoites were grown for 48 hr and transferred to low-serum medium for serum
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starvation. Cells were removed at different time points. Total RNA from ∼5×106 cells was
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purified using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Poly
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A+RNA was isolated using poly A Tract mRNA isolation system (Promega) as per
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manufacturer’s protocol. For northern analysis 10-15 µg of total RNA was resolved on 1.2%
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formaldehyde agarose gel in gel running buffer [0.1 M MOPS (pH 7.0), 40 mM sodium acetate,
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5 mM EDTA (pH 8.0)] and 37% formaldehyde at 4 V/cm. The RNA was transferred on to
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GeneScreen plus R membrane (Perkin Elmer). α- P32dATP labeled probe was prepared by
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random priming method using NEBlot kit (NEB). Hybridization and washing conditions for
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RNA blots were as per manufacturer instructions.
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2 . 3 . L u c i f e ra s e r e p o rt e r c o n s t ru c t a n d s t a b le t r a n s f e c t i o n
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Upstream sequence (900bp) of RPS19 and RPL30 genes were amplified from genomic DNA of
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E. histolytica by PCR using primers AJF and AJR2, and ALF and ALR1 (all primers used in this
131
study are listed in Supplementary Table 1). Primers ALF4 and ALR1 were used for amplifying
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the wild type (244bp) upstream sequence of RPL30 gene for luciferase expression. To mutate the
133
motif1 (Mut 1) an amplicon of 219bp was obtained with primers ALF4 and ALR2M and an
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amplicon of 151bp was obtained with primers ALF5 and ALR which overlap by 27bp. (Primers
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ALR2M and ALF5 contained the mutated sequence). The two amplicons were stitched and a
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344bp fragment was obtained with ALF4 and ALR primers. Finally, using this fragment as a
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template, a 244bp fragment was amplified by using primers ALF4 and ALR1. A schematic
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description is given in supplementary Fig. S3. To mutate the motif 3 (Mut 2) of RPL30, a 244bp
139
fragment was amplified by using primers ALF4 and ALR3 ACC (latter contained the mutated
140
motif 3). To mutate the 5’UTR motif (Mut 3), a 244 bp fragment was amplified by using primers
141
ALF4 and ALR MUT (latter contained mutated 5’UTR). Constructs were cloned upstream of
142
LUC gene at XhoI/Acc651 site in pEh-NEO-LUC vector. Constructs were transfected by
143
electroporation and maintained in presence of G418 at 10µg/ml [14].
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2.4. Luciferase assay
145
This was done as described previously [15]. Briefly, stably transfected trophozoites, maintained
146
in TYI-S-33 medium supplemented with 10 μg/ml G-418, were chilled on ice, harvested and
147
washed twice in 1XPBS (pH 7.4), and lysed in 200 μl of reporter lysis buffer (Promega) with the
148
addition of protease inhibitors E64-C and leupeptin. Lysates were frozen overnight at −80 °C.
149
After thawing on ice for 10 min, cellular debris was pelleted, and the samples were allowed to
150
warm to room temperature. Luciferase activity was measured according to the manufacturer's
151
instructions (Promega) using a Turner Luminometer (model TD-20E). Luciferase activity per
152
microgram of protein was calculated as a measure of reporter gene expression.
153
2.5. Prime r e xtension
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DNase I (Roche)-treated PolyA+ RNA (2µg) was used for primer extension with end labeled
155
oligonucleotides (listed in Supplementary Table 1). Reverse transcription was carried out using
156
superscript III (Invitrogen) at 45°C according to manufacturer's instructions. Sequencing reaction
157
was run on 6% denaturing polyacrylamide gel. The gel image was produced using a Typhoon
158
phosphor imager (GE Healthcare).
159
2.6. Search for the motifs in upstream region of RP genes
160
The online tool MEME [16] was used for prediction of the motifs present in upstream sequence.
161
100 bp of sequence upstream of the RP’s were extracted from E. histolytica HM-I:IMSS genome
162
and were analysed for the motifs. The input consisted of 188 FASTA formatted sequences of
163
RPs with the default settings of width (minimum six and maximum 20) and the search was
164
optimized for identifying zero or one motif for sequence and motif searched for given strand
165
only. For control we used 150 bp upstream sequence to CDS of all E. histolytica HM-I:IMSS
166
8333 genes using Perl- coding. The motifs were searched in these sequences using CisFinder
167
with default parameters except the search was done for forward strand only and maximum
168
number of motif to find were set as 100 (minimum for CisFinder).
169
3. Results
170
3.1. Ribosomal protein transcripts persist during serum starvation.
171
Previous work had shown that unprocessed pre-rRNA accumulates in E. histolytica cells
172
subjected to growth stress due to serum starvation [5]. We were interested to investigate whether
173
transcripts of RP genes also persist during serum starvation in E. histolytica.
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The small (40S) and large (60S) ribosomal subunits are individually made by precise assembly
175
of RPs and rRNAs. The synthesis and turnover of RPs is highly controlled to generate equimolar
176
amount of all RPs [19, 20]. We selected three genes each from the small and large ribosomal
177
subunits (RPS6, RPS3, RPS19, and RPL5, RPL26, RPL30) based on the stage (early, mid or
178
late) at which the protein is thought to be assembled in the newly-forming ribosome [21].
179
Transcript levels were measured in normal and serum-starved cells, by northern hybridization
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with total RNA (Fig. 1). Since each of these genes is present in multiple copies in E. histolytica,
181
the accession number of the copy selected for this study has been indicated in Fig. 1. Multiple
182
copies of the same gene have 97 - 100% amino acid sequence identity and >92% nucleotide
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sequence identity. Each gene probe hybridized with a single band of size expected from the
184
predicted ORF, since UTRs in E. histolytica are generally very short [22]. The transcript levels
185
of none of the six RP genes changed significantly during serum starvation. These data suggest
186
that like pre-rRNA, the transcripts of RP genes also persist at normal levels during serum
187
starvation.
188
3.2. Translation of luciferase reporter (driven by RP genes) is down regulated during
189
serum starvation.
190
Genes encoding the components of translational apparatus are often the targets of post-
191
transcriptional regulation [23]. Since mRNA levels of RP genes did not decline during serum
192
starvation, we were interested to investigate whether translation of these mRNAs continues
193
during serum starvation. For this, we cloned 900bp of the 5’-upstream sequence (upstream from
194
the first AUG codon) of two of the six RP genes studied above (RPS19 and RPL30) to drive a
195
luciferase reporter gene, using Eh-Neo-Luc vector. (The 900 bp upstream sequence included, in
196
addition to the intergenic region, the upstream gene sequence at each locus as well). Stable
197
transfectants were obtained by G418 selection and the levels of luciferase transcripts were
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measured by northern hybridization in cells undergoing serum starvation. As observed for the
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RPS19 and RPL30 transcripts, luciferase transcripts driven from these promoters also persisted
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in starved cells and there was no significant change in transcript levels (Fig. 2 a, b). Further,
201
transcript levels were measured after serum replenishment and, again, no significant change was
202
observed (Fig. 2 c, d). To check whether mRNA translation might be affected during serum
203
starvation, luciferase enzyme levels were measured in cells after shifting to low-serum medium.
204
The data showed that in both cell lines the luciferase activity dropped steadily during serum
205
starvation. After 24 hr of serum starvation the activity was only 7.8% of control in the RPS19
206
cell line and 15% of control in the RPL30 cell line (Fig. 2 e, f). To show that this drop in activity
207
was not due to general instability of luciferase in serum starved cells, we expressed the luciferase
208
gene from the amebic lectin promoter and measured activity during serum starvation. Luciferase
209
activity remained unchanged in these cells (Supplementary Fig. S1). From this we can conclude
210
that translation of reporter luciferase transcript driven by RPS19 and RPL30 upstream sequences
211
is down regulated during serum starvation. We further measured luciferase activity in starved
212
cells after serum replenishment. The data showed that in both cell lines the luciferase activity
213
started going up immediately on serum replenishment and reached almost normal levels by 6 hr
214
(Fig. 2 g, h). Since the luciferase transcript levels in these cells had remained unchanged (Fig. 2
215
c, d), this again confirms that luciferase expression driven by RPS19 and RPL30 upstream
216
sequences is regulated post-transcriptionally.
217
3.3. Analysis of conserved regulatory motifs in 5’ upstream sequences
218
Translational regulation of mRNAs is most frequently mediated by sequences in the 5’UTR. We
219
wished to identify conserved sequence motifs in the upstream sequences (upstream of the
220
initiating AUG codon) of all E. histolytica RP genes, and test whether any of these may be
221
involved specifically in down regulation of RP mRNA translation, without affecting transcription
222
of the gene. We took 100bp of upstream sequences from all RP genes and searched for
223
conserved motifs using MEME motif finder tool. Three conserved motifs were predicted
224
(Supplementary Table 2). Motif 1was found in 75% of RP genes; motif 2 in 50% of RP genes;
225
and motif 3in 69% of RP genes. These motifs were searched in upstream sequences of all E.
226
histolytica genes. Motif 1 was present in 25%, motif 2 in 33%, and motif 3 in 53% of all genes.
227
Thus, only motif 1 was significantly enriched in RP genes.
228
We selected the RPL30 gene to study the effect of mutations in these motifs on transcription and
229
translation. The luciferase reporter system was used for the purpose. Of the three conserved
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motifs the RPL30 gene contained motif 1 (nt position -52 to -58), and motif 3 (-24 to -29) (Fig. 3
231
a). Motif 2 was not found. The transcription start site (TSS) of this gene was mapped by primer
232
extension using a primer from position +79 to +99 with respect to AUG. The TSS was located at
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nt position -29, with respect to AUG (Supplementary Fig. S2). This coincided with the 5’ end of
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motif 3. Mutations were generated as follows (Supplementary Fig. S3). Part of motif 1,
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GAACCC, was mutated to AGGTTT (Mut 1); and motif 3, GAACTT, was mutated to AGGTCC
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(Mut 2). Constructs were generated with luciferase gene cloned downstream of the two mutated
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sequences, and luciferase expression was measured in stable transfectants grown under normal
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and serum-starved conditions. Mut 1 resulted in a severe transcription defect and the level of
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luciferase transcript dropped to ~15% of WT under normal growth conditions, with further
240
decrease during serum starvation (Fig. 3 c, f). Transcription of luciferase reporter was less
241
affected in Mut 2 than Mut 1, but was only~45% compared with WT. These transcripts persisted
242
during serum starvation, with slight decrease at 24 hr as seen in WT cells (Fig. 3 d, f). Luciferase
243
activity was very low in Mut 1 cells, as expected, and in Mut 2 cells it was ~25% of WT under
244
normal growth conditions (Fig. 3 g). When cells were subjected to serum starvation, luciferase
245
activity steadily declined in WT cells. In Mut 2 cells the activity declined initially, but went up to
246
the level in unstarved Mut 2 cells (Fig. 3 g). Since this activity was comparable to that in 24 hr-
247
starved WT cells, it is not possible to comment whether motif 3 may have a role in translational
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down regulation during starvation. However mutations in this motif did have a negative impact
249
on transcription and/or stability of the transcript.
250
3.4. The sequence immediately upstream of AUG (-2 to -9) is involved in translational down
251
regulation during stress
252
To understand the mechanism by which translation of RP genes is controlled during stress, one
253
would need mutants which are specifically affected in translation but show normal transcription,
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which was not the case in the mutants described above. It has been reported in Arabidopsis and
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yeast that sequences immediately upstream of AUG are important modulators of translation
256
efficiency [24, 25]. To test whether the same may be true in the RP genes of E. histolytica, we
257
generated a mutation (Mut 3) at position -2 to -9 just upstream of the AUG in RPL30 gene where
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the WT sequence TGCCAAAG was changed to CATTGGGA. (The -2 to -9 WT sequence is
259
identical in both copies of the RPL30 gene). The mutation was generated as described in
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Supplementary Fig. S3. Unlike previous constructs in which 900 bp of upstream sequence had
261
been used, the Mut 3 construct contained 244 bp of upstream sequence which covered the entire
262
intergenic sequence of 242 bp, but excluded the upstream gene. This mutant had normal levels of
263
luciferase transcripts in unstarved cells (Fig. 3 e, f), and the transcripts persisted at the same
264
levels till 16 hr of serum starvation, after which there was a slight decline in both mutant and WT
265
cells. The luciferase activity in these cells was comparable to WT in unstarved cells, but unlike
266
WT cells it remained high even after 24 hr of serum starvation (Fig. 3 g). Thus the sequence -2 to
267
-9 in the 5’ UTR of the RPL30 gene is required to repress translation of RPL30 mRNA in serum
268
starved cells, and mutations in this region relieve repression.
269
Taken together our data show that during serum starvation E. histolytica blocks ribosome
270
biogenesis post-transcriptionally by inhibiting pre-rRNA processing on the one hand, and the
271
translation of RP mRNAs on the other. Translational inhibition of RPL30 mRNA is controlled
272
by sequences immediately upstream of AUG.
273
4. Discussion
274
The transcription of rRNA and RP genes slows down or stops during growth stress in most
275
model organisms [26, 3]. We have earlier shown that in E. histolytica unprocessed pre-rRNA, in
276
fact, accumulates to high levels during serum starvation [5]. Here we show that transcripts of
277
genes encoding both the small- and large-ribosomal subunit proteins also persist during serum
278
starvation, suggesting that the early branching protist E. histolytica has adopted a post-
279
transcriptional mechanism to regulate ribosome biogenesis whereby it stores pre-rRNA in an
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unprocessed state and RP mRNAs in an untranslated state during growth stress. Such a
281
regulatory mechanism would provide the stressed E. histolytica trophozoite with a large stock of
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RNA intermediates, to be rapidly utilized for assembly of mature ribosomes when conditions are
283
favorable for growth. It is possible that reinitiation of transcription from a silenced template may
284
be a slow process in Entamoeba and therefore gene copies are kept transcriptionally active even
285
when growth slows down.
286
Our data clearly show translational down regulation of RPS19 and RPL30 genes in starved cells.
287
Each of these genes is present in two copies in E. histolytica. The copies of RPS19 show 98%
288
identity and those of RPL30 show 97% identity at amino acid level. The upstream sequences of
289
the two gene copies in each case have no similarity as they occupy different genomic locations.
290
It is, therefore, possible that each copy is subject to differential translational regulation.
291
However, our further analysis of RPL30 showed that mutations of upstream sequence motifs
292
adversely influenced transcription, while the 5’UTR sequence, which is identical in the two
293
RPL30 gene copies, was involved in translational regulation. The 5’UTR of mRNAs is typically
294
one of the most important determinants to modulate translation efficiency. Both in vertebrates
295
and yeast many of the translationally controlled genes have longer 5’UTRs which may form
296
stable stem-loop structures that impede scanning and resist unwinding by translation initiation
297
factors [27]. Sequence-specific binding of trans-acting factors to secondary structure elements in
298
the 5’UTR would inhibit translation [28], which has been well-documented for ferritin mRNA
299
[29]. It is possible that a trans-acting factor binds to the 5’UTR during serum starvation and
300
inhibits translation of the RPL30 gene of E. histolytica. If such an interaction is sequence-
301
specific one may expect to find conserved motifs in 5’UTR and upstream sequences of all RP
302
genes to enable their coordinate control. We found only one upstream motif (motif 1) that could
303
be considered specific to RP genes as it was present in most of them, and was infrequent in other
304
E. histolytica genes. However, at least in the case of RPL30 gene this motif was involved in
305
transcription, rather than translational control. On the other hand we found the involvement of a
306
sequence immediately upstream of AUG in translational control of the RPL30 gene. The
307
importance of nucleotides immediately upstream of AUG in translational regulation has been
308
revealed from studies with Arabidopsis [24] and yeast [25]. In Arabidopsis it was shown that
309
translational efficiency of genes varied over 200-fold depending on their 5’UTR sequences, and
310
the most critical positions were -5 to -1. A-residues at these positions favoured translation while
311
T-residues were repressive [24]. In yeast the positions -3 to -1 upstream of AUG were the most
312
important, and here too the favoured nt was A. Our data show that in E. histolytica the sequence
313
-9 to -2 upstream of AUG in the RPL30 gene was crucial in translational regulation during serum
314
starvation. Both the WT sequence (TGCCAAAG) which repressed translation, and the mutant
315
sequence (CATTGGGA) which relieved the repression, contained A residues at different
316
positions. The contribution of each of these nucleotide residues to translational repression needs
317
to be determined. A search of the -9 to -2 WT sequence (TGCCAAAG) of RPL30 did not reveal
318
identical motifs in 5’UTRs of other RP genes. It is possible that the regulatory sequence motif
319
may be very short, or that the secondary structure of the 5’UTR rather than its sequence may be
320
the important determinant.
321
A common mechanism of translational regulation of genes involved in ribosome biogenesis
322
exists in vertebrates. The mRNAs of all RP genes and other genes coding for components of the
323
translational apparatus contain a 5’terminal oligo pyrimidine (TOP) motif [30]. TOP mRNAs
324
typically initiate with a C-residue followed by an uninterrupted stretch of 4-14 pyrimidines.
325
These mRNAs are translationally repressed under unfavourable growth conditions, and exist as
326
mRNPs. Apart from vertebrates TOP mRNAs have been reported in Drosophila melanogaster,
327
but have not yet been reported in other model organisms like Caenorhabditis elegans and yeast
328
[30]. Thus it is not clear whether this elegant control mechanism is universal. Since the TSS of E.
329
histolytica RP genes has not been mapped it is not possible to determine whether the TOP motif
330
exists in this organism. Our primer extension mapping of RPL30 transcript showed the TSS to be
331
located 29 nt upstream at a G residue, which is incompatible with a TOP motif. We also looked
332
for poly pyrimidine stretches starting with C in the region -10 to -100 upstream of all RP genes.
333
This sequence should include the TSS of most genes as the 5’UTR of E. histolytica genes is
334
<100 nt [22].
335
Post-transcriptional control of gene expression has been reported in other human parasites. In
336
Plasmodium the CDPK1 gene translationally activates repressed mRNA species in the
337
developing zygote, and translation of these mRNAs is dependent on expression of CDPK1 [31].
338
Both the 5’ and 3’ UTRs are required for activation. Genes coding for proteins of the LCCL
339
complex in Plasmodium are transcribed but translationally repressed in gametocytes [32]. In
340
Leishmania the expression of Hsp83 gene is controlled post-transcriptionally through a poly
341
pyrimidine-rich element in the 3’UTR [33]. In Leishmania major, the RPL19 protein is
342
undetectable in stationary phase promastigotes and amastigotes, despite high transcript levels
343
[34]. Studies with the sequences flanking the E. histolytica RPL21 gene also showed that the
344
gene is regulated post-transcriptionally [35], although the sequences involved were not
345
identified. Our study documents the involvement of sequences immediately upstream of AUG in
346
translational regulation in E. histolytica. Such a mechanism has not been reported before, and it
347
will be interesting to know whether this may be a common regulatory mechanism controlling the
348
expression of ribosomal biogenesis genes in E. histolytica.
349
Competing interest
350
The authors declare that they have no competing interests.
351
Author’s contributions
352
SB proposed and designed the research, drafted the final version of the manuscript; AB designed
353
and analyzed the computational work. JA performed the computational and experimental work.
354
SO helped in Northern blotting. AS helped in computational analysis. All authors have
355
participated in preparing the manuscript. All authors have read and approved the final
356
manuscript.
357
Acknowledgements
358
This work was supported by a grant from the department of Science and Technology, India to SB
359
and fellowship from University Grants Commission, India to JA, SO and AS.
360
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Fig. 1. Transcript levels of RP genes during serum starvation. (a & b) Total RNA was isolated at different time points during serum starvation and 15 µg RNA of each sample was electrophoresed (as described in Fig. 1a), blotted and hybridized with full length probes of indicated SSU and LSU RP genes (early, mid and late stage according to the time at which the protein gets assembled in the newly synthesized ribosome). The expected size of each transcript is indicated on the right. (c & d) The band intensity in northern blots was determined by densitometric scanning, and values were normalized with respect to loading control (18S rRNA) in each lane. The data is an average of three independent experiments. N, unstarved cells.
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Fig. 2. Levels of luciferase transcript and luciferase activity in RPS19 and RPL30 cell lines during serum starvation and replenishment. 5’-upstream sequences (900bp) of the RPS19 and RPL30 genes were amplified from genomic DNA of E. histolytica. Both the fragments were cloned at XhoI and Acc651 sites in pEhNeo-Luc vector. Stably transfected cells were grown for 48 hr and subjected to serum starvation for 24 hr, followed by serum replenishment. (a-d) Transcript levels were measured by northern hybridization with full length luciferase probe. (eh) Luciferase activity per µg of protein was measured by luciferase assay. Data presented is of three independent measurements.
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Fig. 3. Mutational analysis of conserved motifs in 5’upstream regulatory sequence of E. histolytica RPL30 gene. (a) The location of conserved motifs 1 and 3 in the RPL30 upstream sequence are indicated, along with their mutated sequences. The sequence of Mut 3 is marked. (b-e) Northern blot analysis of luciferase expressed from WT and mutant RPL30 sequences in serum starved (SS) cells. (f) Luciferase transcripts in unstarved mutant cell lines relative to WT, (‘N’ lanes in b-e) and (g) luciferase activity in WT and mutants during serum starvation.
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Fig. 1
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Fig. 2
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Fig. 3