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The U6 snRNA-encoding gene of the monogenetic trypanosomatid Leptomonas collosomat
Gene, 156 (1995) 139-144 © 1995 Elsevier Science B+V. All fights reserved. 0378-1119/95/$09.50
139
GENE 08766
The U6 snRNA-encoding gene of the monogenetic trypanosomatid
Leptomonas collosoma? (Spliceosome; trans-splicing; spliced leader RNA; U2 RNA; U6 flanking sequence; tRNA)
Anat Goldring and Shulamit Michaeli Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel Received by A. Kohn: 18 July 1994; Revised/Accepted: 18 September/1 November 1994; Received at publishers: 13 January 1995
SUMMARY
The U6 snRNA (U6) is the most conserved small nuclear RNA (snRNA) and apparently plays a central role in catalysis of the cis-splicing reaction. In trans-splicing, U6 may have an additional function. In the nematode transsplicing system, a direct interaction between the U6 and spliced leader (SL) RNAs has been demonstrated, suggesting that U6 may serve as a bridge between the SL RNA and the acceptor pre-mRNA. To examine possible phylogenetic conservation of trypanosomatid U6 sequences that may interact with spliceosomal RNAs, we have cloned and sequenced the U6 gene from the monogenetic trypanosomatid Leptomonas collosoma (Lc). The Lc U6 deviates from the Trypanosoma brucei (Tb) RNA only in four positions located in the 5' stem-loop and the central domains. As in Tb, U6 is a single-copy gene and two tRNA genes, tRNA m" and tRNAn% are found upstream to the gene. The tRNAs are differentially expressed; tRNA al" is transcribed in the opposite direction to U6, whereas tRNA It~ is not transcribed. Possible base-pairing between U6 and the U2 and SL RNAs, similar to the interactions that take place in the nematode trans-splicing system, are proposed.
In trypanosomatids all mRNA contain a 39-nt spliced leader (SL) at the 5' end. The SL sequence is acquired from a small RNA, the SL RNA, by trans-splicing. Trans-splicing is analogous to cis-splicing, as it proceeds through two transesterification steps creating a Y-structure intermediate, analogous to the lariat of the cis-splicing reaction (Agabian, 1990). Trans-splicing, like cis-splicing, takes place on a RNP particle, which has
been shown to contain U snRNPs and the unique particle, SL RNP (Michaeli et al., 1990; Cross et al., 1991). U2, U4 and U6 have been shown to actively participate in trans-splicing (Tschudi and Ullu, 1990). Although the trypanosome U snRNAs are related in sequence to the cis-splicing homologues, they possess several unique features (Mottram et al., 1989). None of these small RNAs contain the Sm-binding site or bind Sm antigens (Michaeli et al., 1990; Palfi et al., 1991). Apparently, no U! or U5 exists in trypanosomes (Mottram et al., 1989),
Correspondence to: Dr. S. Michaeli, Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel. Tel. (972-8) 343 626; Fax (972-8) 468 256; e-mail: [email protected] #We wish to dedicate this paper to the memory of Professor Alexander Kohn (known to his friends as Leshek), a pioneer in the development of live viral vaccines, founder of the Journal of lrreproducible Results and a leading figure in the Israeli scientific community. Leshek strove for honesty in science, but did so ~vith a great sense of humor. We will all miss him.
Abbreviations: AMT, aminomethyltrioxsalen; bp, base pair(s); C., Crithidia; Cf C. fasciculata; kb, kilobases(s) or 1000 bp; L., Leptomonas; Lc, L. collosoma; Ls, L. seymouri; N, A or C or G or T; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PSE, proximal sequence element; R, A or G;; RNP, ribonucleoprotein; SDS, sodium dodecyl sulfate; SL, spliced leader; Sin, antigens against which a patient (Smith) with lupus syndrome made antibodies; snRNA, small nuclear RNA; SSC, 0.15 M NaC1/0.015 M Na~.citrate pH 7.6; T., Trypanosoma; Tb, T. brueei; TMG, trimethylguanosine; U6, U6 snRNA; U6, gene encoding U6; U snRNA, uridylic-acid-rich snRNA; UV, ultraviolet; Y, C or T.
INTRODUCTION
SSDI 0 3 7 8 - 1 1 1 9 ( 9 5 ) 0 0 0 4 8 - 8
140 and it has been suggested that the SL RNA may have a dual function serving both as a substrate as well as a Ul-like snRNA (Bruzik et al., 1988). Recently, a novel RNA termed spliced leader associated RNA (SLA) was identified in Trypanosoma brucei (Tb) and was proposed to be the trans-spliceosomal U5 homologue (Watkins et al., 1994). There are still many open questions regarding the mechanism of trans-splicing. For example, it is not known how the SL RNA interacts with the pre-mRNA during the trans-splicing reaction. The phenomenon of transsplicing has now been extended to other systems (Bonen, 1993), the nematodes being the most studied one (Nilsen, 1993). Recently, in a functional trans-splicing assay using aminomethyltrioxsalen (AMT)-dependent UV crosslinking, a direct interaction between the nematode SL RNA intron sequence and the U6 RNA was demonstrated (Hannon et al., 1992). The great interest in the U6 RNA originates from the many observations supporting the notion that U6 has a catalytic function in splicing (Wise, 1993). U6 is the most conserved spliceosomal U snRNA in both length and sequence (Brow and Guthrie, 1988). The hexanucleotide 5'-ACAGAG, located in the central domain of the RNA, is found in all U6 RNAs sequenced so far, and mutations in this region block the first or the second steps of the splicing reaction (Fabrizio and Abelson, 1990; Madhani and Guthrie, 1992; Datta and Weiner, 1993). Recent experiments provide evidence for an intimate association of the ACA sequence with the 5' splice site (KandelasLewis and S6raphin, 1993; Lesser and Guthrie, 1993), suggesting that the 5' splice site is delivered to the catalytic center by pairing with the U6 RNA. To identify potential features specific for trans-spliceosomal U6 RNA, we have cloned and sequenced the U6 gene from the monogenetic trypanosomatid Lc. Using this data and recent sequence data that became available from other trypanosomatids (Xu et al., 1994), we propose possible base-pairing between the SL and the U6 RNA, similar to the interaction that takes place in nematodes. To elucidate the regulatory elements that may control the transcription of the U6 gene, we have analyzed the upstream sequence and revealed two tRNA genes which are differentially expressed and may serve as extragenic promoter elements of the gene.
EXPERIMENTALAND DISCUSSION (a) G e n o m i c organization of the U6 gene-linkage to two t R N A genes
To determine the copy number of the Leptomonas collosoma (Lc) DNA was Southern analysis with the homologous probe (Fig. 1). A single hybridization band
Lc U6 gene, subjected to Tb plasmid was observed
upon digesting genomic DNA with either an enzyme of a 6-bp recognition site (PvulI) or with enzymes of a 4-bp recognition site (Sau3A and TaqI), suggesting that the U6 is a single-copy gene. The sequence of the U6 gene, including 471 nt upstream and 392 nt downstream derived from HpalI subclones, is presented in Fig. 2. Since two genes (for tRNA TM and tRNA TM)were found upstream to the Tb U6 gene (Mottram et al., 1991), we examined whether such an arrangement also exists for the Lc U6 locus. Indeed, two tRNA genes were found, a Gin and a tRNAtuAA~ lie t R NAtctm) which have a transcriptional configuration identical to that of the Tb tRNAs. The sequence of the tRNAs and their inferred secondary structure is presented in Fig. 3. The box A of tRNA Ile is 5'-TAGCTCAGTCGG, which deviates from the canonical T R R Y N N A G T G G sequence by an additional C, whereas box B, 5'-GTTCGACCCC, agrees well with the consensus G T T C R A N N C C sequence (Geiduschek and Tocchini-Valentini, 1988). Also the box A of tRNA aln (5'-TAGTGTAGCGG) deviates from the consensus, whereas box B is 100% conserved. Similar to Tb Mottram et al., 1991) the distance between the first tRNA and the U6 gene is longer (99 nt) compared to the dis-
,,< kb
~
31_946 ::i !i:iii! 4.3
i li
i
2.3 m 2.0 m
0.5 m
Fig. 1. Southern analysis of Lc U6 sequence. Lc (Nelson et al., 1984) was provided by Dr. Nina Agabian (University of California, San Francisco, CA, USA). Genomic Lc DNA (10 ~tg) was digested with PvuII, Sau3A and Taql, separated on a 1.0% agarose gel, and transferred to Nytran membrane. The membrane was hybridized with the random-labelled Tb U6 gene probe. The Tb probe was a pGEM-3 vector carrying a 342-nt HaellI fragmentfrom the U6 locus (Mottram et al., 1989). Hybridization was at 65°C in 5xSSC/0.1% SDS/5× Denhardt's solution/100~tg per ml salmon sperm DNA, and the probe was washed by 0.1 x SSC at 65°C. Molecular mass markers (given in kb) are ~. DNA digested with HindllI.
141 •
-471
°
.
.
°
°
G G A C G C T G G G T A A A A C A C CAC G A A A ~ C G G A A A G T G G G G T A G C T G G G G T T C C C G A G T T G
-411
G TCAAAGGGGCAAGACTTAAGTTCTTGTGC GCAACGTTCGTGGGTTCGAACCCCACCTCC
-351
AGCATAGTTTTC C CTTTCC GCCGACTGGACATGTACCATGAAAGTCGCAGCGGCTGCCGA
-291
AGCTCTCATAGCTCAGTC GGTTAGAGCGTGGGTCTAATAAG CCCAAGGTCACAGGTTCGA
-231
C C C C T G T T G G G A G C A C T T T T C CAATC G C A C G T T T T T T C T T T T C G C G A C G G G A G A A A A A G T
-171
CACTCCTACCTGGACTCGAAC CAGGGTTATCGGATTCAGAGTCCGAGG TGATAACCGCTA
-111 -61
C A C T A T A G G A G C G C A C G C C T G T G C T G G C G TGGGC G T A G T T G T A C T T G C T A A T T C A C A A T A +1 C T A A G A T A C G G A A A A C A C G T G T C A C T G C G A A C G T T T T T T C C T C T A A G A A G C G G A G CC C C T
+i0
T C G G G G G A .CA,T C C A C A A C C TG ,,GAACTTCAACAGAGAAGATTAGCAC TC T C C C T G C G C A A G
+70
GCTGATGTCAATCTTCGAGAGATATAG CTTTTCGCCACGCTTTCCTCAC GTGTTTTCCCT
-371 t g g g t t c g a a c c ' c c a c c ~ c c a g c a t a g t ttt cc'ct tt c c g c c g a c t g g a c a t g t a c c a t g Ile tRNA -311 a a a g t c gc agc g g c t g c c g a a G C T C T C A T A G C T C A G T C G G T T A G A G C G T G G G T C T A A T A A -251 G C C C A A G G T C A C A G G T T C G A C C C C T G T T G G G A G C A c t t t t c caat cgc acgt t t t t t c t t box B -191 t t c gc ga c g g g ag a a a a a g t CACTCCTACCT~C~ACTCO.AACCAGGGTTATCGGATTCAGA GI.tR~A box B -131 O T C C G A G G T G A T A A C C G C T A C A C T A T A G G A ~ C g c a c g c c t gt gct g g c g t g g g c g t agt t box A -71 gtacttgctaattcacaatactaagatacggaaaacacgtgtcactgcgaacgttttttc -II c t c t a a g a a g c
Gln tRNA
* 130
TCAG T G G C T T A G A A G A C T A G T T C A C C G G A C G A G A G A G A T G G A G A G A G A G A G A G A A G C T G C
+190
C G C A C G G C T T G T G T G C G G G T T C C C C C G C C C C C C A C C C T C A C TCC C A A G C T C A C C G CGCAC
+250
CGTTTTCGGCGCGGGGGAGGGAGGGGTGCGCACCGCCCGACGGCGCCCGCCGCGAGCCCT
*310
TCC CCC CAC G C C C C A C GCG GG GCAAAC C CAAC C C A G C A C G C G C A C A C CACCC C A G G G A G A
4-370 C C C C G C C A G G C C C C C T C T A T C T T
392
Fig. 2. Sequence of the Lc U6 gene. The U6 gene was cloned from a kEMBL3 library carrying 15-20-kb Sau3A genomic fragments (Goldring et al., 1994) that was screened with the U6 plasmid probe (Mottram et al., 1989). Two positive clones carrying overlapping genomic segments were further used to subclone the gene. Fragments generated from digesting the phages with either TaqI, HpaII or Sau3A were subcloned into M13 and pGEM-3 vectors at the AccI and BamHI sites, respectively. Sequence analysis was performed using the dideoxy chaintermination method (Sanger et al., 1977) using flanking vector primers or the following synthetic oligos. Oligo #5958 is coding for nt + 136 to + 150, 5'-GGCTTAGAAGACTAG; oligo #6451 is an anti-sense to the tRNA ~j" nt - 162 to - 143, 5'-CTGGACTCGAACCAGGGTTA; and oligo #6452 is sense to #6451. The position of the first nt of the U6 RNA-coding sequence is designated as + 1. The sequence of the U6 RNA (101 nt) is underlined. This sequence data have been submitted to the EMBL/GenBank database with accession No. X79014. tance between the t R N A s (50 nt). Recently, the U6 gene was cloned and sequenced from Leptomonas seymouri and Crrithidiafasciculata (Xu et al., 1994). Identical c o m p a n ion t R N A s were found upstream to these t r y p a n o s o m a t i d U6 genes. As previously p r o p o s e d on the basis of r R N A (Fernandes et al., 1993), the conservation in the U6 c o m p a n i o n t R N A also suggests that Crithidia and Leptomonas are closely related to each other m o r e than each of them to Tb. The distance between the the the first t R N A and the U6 gene is highly conserved (97, 97, 98 and 99 bp in Tb, C f Ls and Lc, respectively), suggesting that this spacing m a y have functional significance. To examine the expression of both the U6 and the c o m p a n ion tRNAs, the entire clone, as well as specific probes c o m p l e m e n t a r y to the U6 and the two tRNAs, were used in N o r t h e r n analysis. The results are presented in Fig. 4, and indicate that at least two R N A transcripts are encoded by the U6 locus. Since the difference between the two t R N A s are only 2 nt, the expression of the individual t R N A s was examined using specific probes for each of the tRNAs. A 285-nt NruI-HindIII fragment, specific for t R N A '~, and an oligo, c o m p l e m e n t a r y to the t R N A G~", were used. The results indicate that t R N A a~" is expressed (Fig. 4C-6) whereas the t R N A n~ is not (Fig. 4D-8). To examine the specificity of the probes used in the N o r t h e r n analysis, Tb R N A was used as a control.
D loop GcGAUGuGAA
G G U CG U A CG C G U A
A G C C G U A C G U G P loop C G CC ,~ A UGAcucGAU A U U G U C C GC cA C A G G U u CG G AGAGC U UU GUAA~G T ~ C loop GC ~ GC GO UG C A U A AAU t R N A II~
U GGAccUGA CCUGGuucG
~A
G U u A U C A C c U AU'" CG GC GC AU C A A UCUG anticodon
T~IJC loop
an~icodon
Fig. 3. The tRNAs upstream to the U6 gene-sequence and their deduced secondary structure. Homology to tRNA molecules was determined by comparing the sequence to sequences in the EMBL data Library, Nucleotide sequence Database, and GenBank release 70 from InteUiGenetics (Mountain View, CA, USA). Sequence analysis software package of the Genetics Computer Group version 7.0 from April 1991 by John R. Devereaux was used to determine the tRNAs structure. The inferred tRNA sequences are indicated by upper-case lettering. Consensus polIII promoter elements (box A and box B) are underlined. The direction of transcription is indicated with an arrow in each case. The predicted secondary-structure models of tRNAG~nand tRNAne are shown. The anticodon, TOC and D loops are indicated.
The hybridization data in Fig. 4A-l, B-3, C-5 and D-7 indicate that in contrast to the U6 gene, the primary sequence of the t R N A s are not very well conserved. Neither the Tb nor the Lc U6 upstream sequence contain elements that resemble the TATA box or the PSE elements found upstream to the m a m m a l i a n U6 gene ( L o b o and Hernandez, 1990). In addition, no internal or downstream box A and box B can be found in the sequence similar to the p r o m o t e r elements of yeast (Brow and Guthrie, 1990). The identification of the U6 prom o t e r awaits functional analysis. However, the close proximity of t R N A s to the U6 suggests that these genes m a y be functionally related, possibly by increasing the local concentration of R N A polymerase III and its specific transcription factors. Four changes were revealed by c o m p a r i n g the Lc to the Tb U6 R N A sequence. All the changes are located in either the 5' terminal loop or the central domain. Two nt additions in position 8 and 13 resulted in an increased 5' stem. Such an increased stem was also revealed in Ls and C f U 6 sequences (Xu et al., 1994). The other changes are A 2s of Tb to C 27 in Lc and A 32 in Tb to C 34 in Lc. In Ls the U27 corresponds to C in C f like in Le. The changes in this position lie within the sequence h o m o l o gous to the A A U U sequence found in almost all U6 RNAs. In Tb the h o m o l o g o u s sequence to A A U U is
142
A
B
I
nt
1
I
2
!
3
D
C
I
4
l !
I
5
6
7
l
8
217-201-190" 180" 160-147-123-ii0--
90-76-67--
Fig. 4. Northern analysis with the U6 and tRNA genes. RNA was extracted from cells using the LiCl-urea extraction procedure as was previously described (Murphy et al., 1986). Total RNA ( 10 gg) prepared from Tb and Lc were resolved on a 7 M urea-6% polyacrylamide gel and electroblotted onto a Nytran membrane. Hybridization with labelled oligo was performed at 37°C in 5x SSC/0.1% SDS 5 x Denhardt's solution/100 gg per ml salmon sperm DNA, and the probe was washed at 37°C with 2 x SSC/0.1% SDS. (A) The Lc U6 HpaII subclone; (B) anti-sense oligo complementary to Tb U6 sequence nt 28-46, 5'-TCTTCTCTGTTGAATTTCC; (C), anti-sense oligo complementary to tRNA ~l" (# 6451 ); (D), a 285-nt (NruI-HindlIl) fragment carrying the tRNA ae. RNA from Tb was in lanes 1, 3, 5, 7 and Lc RNA in lanes 2, 4, 6 and 8. Size markers (pBR322 digested with HpalI) are indicated.
AACU, in Lc and Cf the sequence is ACCU and in Ls AUCU. Interestingly, in the nematodes, mutations within the AAUU sequence resulted in branch formation between the splicing substrate and the U6 (Yu et al., 1993). The sequence upstream to the conserved hexanucleotide ACAGAG is in all trypanosomatids UUCA, as opposed to the concensus sequence [CRA. (where a dot indicates that two or more nt differences were found among the sequences, including deletion or insertion of a residue) or CRAY (after excluding the trypanosomatid U6 sequences from the list)I, found in other U6 RNAs (Guthrie and Patterson, 1988). The significance of these changes, if any, may be understood when more is known about U6 snRNA-pre mRNA interactions during the trans-splicing reaction. (b) The Lc U6 RNA and its potential to interact with the U2 RNA It is currently unknown what types of interactions take place during the trypanosome trans-splicing reaction that involve the U6 RNA. Work done by Watkins and Agabian (1991), demonstrated that the Tb 5' end of the U2 RNA can be cross-linked in vivo to the 3' end of the U6 molecule, anlogons to the U2-U6 interaction in the mammalian system. The cross-linked region was grossly
mapped to be downstream to nt 29 and 70 of the U2 and U6, respectively. In the mammalian system, a similar region (U2, 3-11; U6, 87-95), was proposed on the basis of psoralen cross-linking (Hausner et al., 1990) and by introducing mutations in this region that influence the efficiency of mRNA splicing (Wu and Manley, 1991). Interestingly, this area of interaction in yeast, is fairly tolerant to mutations (Madhani and Guthrie, 1992). A potential for base-pair interaction between the Lc U2 and U6 RNAs is presented in Fig. 5. The proposed interaction domain between the 5' end of the U2 and the 3' end of the U6 RNA is shorter (10 nt) in Lc compared to the structure previously proposed for Tb (15 nt) (Watkins and Agabian, 1991). The length of this interaction domain, therefore, resembles more the domain proposed for mammals (9 nt) (Wu and Manley, 1991) and nematodes (8 nt) (Yu et al., 1993). In addition, the model presented in Fig. 5 includes the possible conserved interaction domain of helix I a and b (Madhani and Guthrie, 1992). Mutations in this domain in yeast impaired both steps of splicing, suggesting that this domain may be involved in catalysis of both reactions (Madhani and Guthrie, 1992). Interestingly, this domain lies just upstream to the branch point interaction region (the GUAGUA box) which is missing from the trypanosomatid U2 RNAs (Mottram et al., 1989; Hartshorne and Agabian, 1990). The potential for simultaneous basepairing between the U2/U6 helix I and a U2/pre-mRNA may therefore be different in trypanosome trans-splicing compared to cis-splicing. (c) Putative SL-U6 interaction in trypanosomatids The recent evidence for interaction of the U6 RNA with the 5' splice site in mammalian and yeast cis-splicing systems, suggests that U6 RNA may replace the U1 RNA in base-pairing with the 5' splice-site region (Wise, 1993). Since in trypanosome trans-splicing the 5' splice site is always located on the SL RNA, one might expect to find a potential for base-pairing interaction between the 5' CGC Gu--AA C~G
cG C-G C-GC_ G
cc-~
U6
~, he]i~ I=~he%"k. U=A U~G
heli~ II
Fig. 5. Potential interactions of the Lc U2 and U6 RNA. The U2 sequence is from Hartshorne and Agabian (1990). The model includes the potential interaction in helix I a and b, according to (Madhani and Guthrie, 1992). The interaction in domain II is in agreement with the interaction domain suggested for mammals (Hausner et al., 1990) and nematodes (Yu et al., 1993) and is different from the structure proposed for Tb (Watkins and Agabian, 1991).
143 splice site region of the SL RNA and the conserved hexanucleotide ACAGAG. Indeed, inspection of all trypanosomatid SL RNA sequences indicate conservation of the UG sequence at position + 4 and + 5 that can pair with the first conserved nucleotides AC. Lc is the only trypanosomatid SL RNA in which SL RNA possesses the sequence UGU that can potentially interact with the entire ACA sequence. Interestingly, the Cf U6 RNA carries a C ~ T change at the second position of the ACAGAG sequence and a compensatory base change of the corresponding SL RNA at position + 5, supporting the notion that the 5' splice site/U6 RNA interaction may be shared between cis- and trans-splicing systems (Xu et al., 1994). The U6/5' splice site is believed to be one of the last interactions before catalysis. One may, however, expect to find interactions between these molecules that take place at earlier stages of the reaction. Such an interaction was indeed revealed in the nematode system between the 3' end of the U6 sequence and the region of the Sin-binding site in the SL RNA (Hannon et al., 1992). The SL/U6 interaction permits simultaneous basepairing of the U6 RNA with both the SL and U2 RNA, suggesting that the U6 and U2 may serve as a connecting bridge between the SL RNA and the pre-mRNA (Hannon et al., 1992). To examine whether such interactions may also take place in trypanosomes, we searched for potential base-pairing interactions between the U6 and the trypanosomatid SL RNA molecules. Such a putative interaction (presented in Fig. 6), was revealed by searching for a U6 RNA sequence complementary to the 'Sm-like' binding site, located between stem-loops II and III of the trypanosomatid SL RNA. The U6 sequence of all trypanosomatids tested is identical in the proposed interaction domain (nt 68-86 in Tb and nt 70-88 in the other trypanosomatids), whereas the SL RNA sequence in this region is different, except for the sequence U(3_41GGR, which is common to all trypanosomatid SL RNAs. The putative interaction domain between the Tb molecules is extensive and is composed of 16 nt, whereas in Leptomonas sp. and Crithidia only the first 6-8 nt have the potential to interact. In nematodes it has been demonstrated that despite the extensive potential for basepairing interaction of 19 nt between the SL and U6 RNA (shown in Fig. 6), only 11 nt most probably compose the interaction domain (Hannon et al., 1992). It has been long demonstrated that no Sm antigens exist in trypanosomes (Michaeli et al., 1990; Palfi et al., 1991) and that the trypanosomatid U snRNAs lack the Sm-binding site (Mottram et al., 1989). The existence of a canonical Sm-like site in the trypanosomatid SL RNA (Bruzik et al., 1988), especially in the Leptomonas and Leishmania sequences, was always puzzling. As elucidated from the nematode study, the Sm-binding site and adjacent four 3' nucleotides are sufficient to confer the SL RNA function (Harmon et al., 1992). The conservation of this
C) SL 3' O H
OH
~x~0 UOUAAACCUUC,C GAAGUG----~ U
Gppp~
6
n ema t ode
\ p~pX
I
113
G
O
13o S L
~,c?, ,7"~,,.~ ,.~9,7c.~,
3.
OH / AGAGCUUCUAACU~GA G U C ~ 6 Gppp
76
k
93
SL ~ GGACCGAGCUUUCGG III I • II,I U 6 AGAGCUUCUAACUGUAGUC 78 CC~
sT. i . ~ l . ~ . o~ ~. ?
,.,
o~
Tb pppX
LC
~.s
U6 AGAC-CU'UCUAACUGUAGUC 73 CG IGIAAGCC~ S L UUUUGAGG I.i.ll.i. " t" U6 AGAGCUUC U CUGU GUC AA A
cf
Fig. 6. Potential for SL and U6 RNA interaction in trans-splicing. The nematode interaction domain is from Hannon et al. (1992). The Sm-binding site of the nematode SL RNA is indicated. The Tb SL RNA and U6 sequences are form Milhausen et al. (1984) and M o n r a m et al. (1989), respectively. The Lc sequences are from (Milhausen et al. (1984) and Goldring et al. (1994). The Ls SL RNA is from Bellofatto et al. (1988) the Cf SL RNA sequence from Muhich et al. (1987). The U6 sequence of C f a n d Ls are are from Xu et al. (1994). The U6 sequence comprising the U6-SL interaction domain is nt 68-86 for Tb and nt 70-88 for all other trypanosomatid species.
domain in trypanosomes and nematodes may be dictated by its absolute requirement to interact with the U6 RNA for catalysis of the trans-splicing reaction. The validity of the proposed trypanosomatid SL-U6 interactions awaits corroborating biochemical evidence for SL-U6 crosslinking in this region. Conclusions (1) The Lc U6 RNA is essentially identical to the Tb sequence apart from four changes, all located in the 5' stem-loop and the central domain. (2) The upstream 5' flanking region of the U6 gene harbors sequences for two tRNA genes which are expressed differently, tRNA ~ln is found 99 nt upstream to the U6 gene and is transcribed in an opposite direction to that of U6 gene. In contrast, tRNA ~1°is located 45 nt upstream to tRNACln; it is encoded by the same strand as the U6 RNA, but it is not transcribed. These tRNA genes are most probably the extragenic elements controlling the transcription of the U6 gene. (3) A potential for base-pair interaction between the 5' end of the U2 RNA and the 3' end of the U6 RNA is proposed. The interaction domain includes helix Ia, Ib and II, which are analogous to the interaction domains in mammals and yeast. (4) Two types of interactions are proposed between the SL and the U6 RNA. One interaction (between positions +4 to +6 of the SL RNA intron (UGU) with the ACA
144 s e q u e n c e o f t h e U 6 h e x a n u c l e o t i d e ) , r e s e m b l e s the intera c t i o n o f the cis-splicing i n t r o n s e q u e n c e at t h e 5'-splicesite j u n c t i o n w i t h the s a m e c o n s e r v e d U 6 h e x a n u c l e o t i d e s e q u e n c e . T h e o t h e r is a trans-splicing-specific i n t e r a c t i o n t h a t i n v o l v e s t h e 3' e n d o f t h e U 6 R N A a n d the S L R N A at the ' S m - l i k e ' b i n d i n g site. T h i s i n t e r a c t i o n is b a s e d o n a c o n s e n s u s s e q u e n c e U t 3 _ a ) G G R , w h i c h is s h a r e d a m o n g t h e t r y p a n o s o m a t i d S L R N A a n d is s i m i l a r to the n e m a t o d e s e q u e n c e U~a)GGA, t h a t was p r e v i o u s l y s h o w n to i n t e r a c t in v i t r o w i t h t h e U 6 s e q u e n c e . ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m the J o h n D. a n d
Catherine
T. M a c A r t h u r
Foundation
to the
W e i z m a n n I n s t i t u t e o f Science. REFERENCES Agabian, N.: Trans-splicing of nuclear pre-mRNAs. Cell 61 (1990) 1157 1160. Bellofatto, V., Copper, R. and Cross, G.A.: Discontinuous transcription in Leptomonas seymouri: presence of intact and interrupted miniexon gene families. Nucleic Acids Res. 16 (1988) 7437-7456. Bonen, L.: Trans-splicing of pre-mRNA in plants, animals, and protists. FASEB J. 7 (1993) 40-46. Brow, D.A. and Guthrie, C.: Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature 334 (1988) 213 218. Brow, D.A. and Guthrie, C.: Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position. Genes Dev. 4 (1990) 1345-1356. Bruzik, J.P., Van Doren, K., Hirsch, D. and Steitz, J.A.: Trans-splicing involves a novel form of small nuclear ribonucleoprotein particles. Nature 335 (1988) 559-562. Cross, M., Gtinzl, A., Palfi, Z. and Bindereif, A.: Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei : structural organization and protein components of the spliced leader RNP. Mol. Cell. Biol. 11 (1991) 5516-5526. Datta, B. and Weiner, A.M.: The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae. Mol. Cell. Biol. 13 (1993) 5377-5382. Fabrizio, P. and Abelson, J.: Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing. Science 250 (1990) 404-409. Fernandes, A.P., Nelson, K. and Beverley, S.M.: Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa. Proc. Natl. Acad. Sci USA 90 (1993) 11608 11612. Geiduschek, E.P. and Tocchini-Valentini, G.P.: Transcription by RNA polymerase III. Annu. Rev. Biochem. 57 (1988) 873-914. Goldring, A., Karchi, M. and Michaeli, S.: The spliced leader RNA gene of Leptomonas collosoma. Exp. Parasitol. (1994) in press. Guthrie, C. and Patterson, B.: Spliceosomal snRNAs. Annu. Rev. Genet. 22 (1988) 387-419. Hannon, G.J., Maroney, P.A., Yu, Y.-T, Hannon, G.E. and Nilsen, T.W.: Interaction of U6 snRNA with a sequence required for function of the nematode SL RNA in trans-splicing. Science 258 (1992) 1775-1780. Hartshorne, T. and Agabian, N.: A new U2 RNA secondary structure provided by phylogenetic analysis of trypanosomatid U2 RNAs. Genes Dev. 4 (1990) 2121 2131. Hausner, T.-P., Giglio, L.M. and Weiner, A.M.: Evidence for basepairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes Dev. 4 (1990) 2146-2156.
Kandels-Lewis, S. and S6raphin, B.: Role of U6 RNA in 5' splice site selection. Science 262 (1993) 2035-2039. Lesser, C.F. and Guthrie, C.: Mutations in the U6 snRNA that alter splice site specificity: implications for the active site. Science 262 (1993) 1982-1988. Lobo, S.M. and Hernandez, N.: A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter. Cell 58 (1990) 55-67. Madhani, H.D. and Guthrie, C.: A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Cell 71 (1992) 803-817. Michaeli, S., Roberts, T.G., Watkins, K.P. and Agabian, N.: Isolation of distinct small ribonucleoprotein particles containing the spliced leader and U2 snRNAs of Trypanosoma brucei. J. Biol. Chem. 265 (1990) 10582-10588. Milhausen, M., Nelson, R.G., Sather, S., Selkirk, M. and Agabian, N.: Identification of a small RNA containing the trypanosome spliced leader: a donor of shared 5' sequences of trypanosomatid mRNAs? Cell 38 (1984) 721-729. Mottram, J., Perry, K.L., Lizardi, P.M., Ltihrmann, R., Agabian, N. and Nelson, R.G.: Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp, brucei: identification of the U2, U4, and U6 RNA analogs. Mol. Cell. Biol. 9 (1989) 1212-1223. Mottram, J.C., Bell, S.D., Nelson, R.G. and Barry, J.D.: tRNAs of Trypanosoma brucei: unusual gene organization and mitochondrial importation. J. Biol. Chem. 266 (1991) 18313-18317. Muhich, M.L., Hughes, D.E., Simpson, A.M. and Simpson, L.: The monogenetic kinetoplastid protozoan, Crithidiafasciculata, contains a transcriptionally active, multi-copy mini-exon sequence. Nucleic Acids Res. 15 (1987) 3141-3153. Murphy, W.J., Watkins, K.P. and Agabian, N.: Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans-splicing. Cell 47 (1986) 517-525. Nelson, R.G., Parsons, M., Selkirk, M., Newport, G., Barr, P. and Agabian, N.: Sequences homologous to variant antigen mRNA spliced leader in Trypanosomatidae which do not undergo antigenic variation. Nature 308 (1984) 665-667. Nilsen, T.W.: Trans-splicing of nematode pre-mRNA. Annu. Rev. Microbiol. 47 (1993) 413-440. Palfi, Z., Gt~nzl, A., Cross, M. and Bindereif, A.: Affinity purification of Trypanosoma brucei snRNPs reveals common and specific protein components. Proc. Natl. Acad. Sci. USA 88 (1991) 9097-9101. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Tschudi, C. and Ullu, E.: Destruction of U2, U4, or U6 small nuclear RNA blocks trans-splicing in trypanosome cells. Cell 61 (1990) 459-466. Watkins, K.P. and Agabian, N.: In vivo UV cross-linking of U snRNAs that participate in trypanosome trans-splicing. Genes Dev. 5 (1991) 1859-1869. Watkins, K.P., Dungan, J.M; and Agabian, N.: Identification of a small RNA that interacts with the 5' splice site of the Trypanosoma brucei spliced leader RNA in vivo. Cell 76 (1994) 171 182. Wise, J.A.: Guide to the heart of the spliceosome. Science 262 (1993) 1978-1979. Wu, J. and Manley, J.L.: Base-pairing between U2 and U6 snRNAs is necessary for splicing of mammalian pre-mRNA. Nature 352 (1991) 818-821. Xu, G-L., Wieland, B., and Bindereif, A: trans-spliceosomal U6 RNAs of Crithidiafasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG seqquence and potential base pairing with spliced leader RNA. Mol. Cell. Biol. 14 (1994) 4565-4570. Yu, Y.-T., Maroney, P, A., and Nilsen, T.W.: Functional reconstitution of U6 snRNA in nematode cis- and trans-splicing: U6 can serve as both a branch acceptor and a 5' exon. Cell 75 (1993) 1049-1059.
139
GENE 08766
The U6 snRNA-encoding gene of the monogenetic trypanosomatid
Leptomonas collosoma? (Spliceosome; trans-splicing; spliced leader RNA; U2 RNA; U6 flanking sequence; tRNA)
Anat Goldring and Shulamit Michaeli Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel Received by A. Kohn: 18 July 1994; Revised/Accepted: 18 September/1 November 1994; Received at publishers: 13 January 1995
SUMMARY
The U6 snRNA (U6) is the most conserved small nuclear RNA (snRNA) and apparently plays a central role in catalysis of the cis-splicing reaction. In trans-splicing, U6 may have an additional function. In the nematode transsplicing system, a direct interaction between the U6 and spliced leader (SL) RNAs has been demonstrated, suggesting that U6 may serve as a bridge between the SL RNA and the acceptor pre-mRNA. To examine possible phylogenetic conservation of trypanosomatid U6 sequences that may interact with spliceosomal RNAs, we have cloned and sequenced the U6 gene from the monogenetic trypanosomatid Leptomonas collosoma (Lc). The Lc U6 deviates from the Trypanosoma brucei (Tb) RNA only in four positions located in the 5' stem-loop and the central domains. As in Tb, U6 is a single-copy gene and two tRNA genes, tRNA m" and tRNAn% are found upstream to the gene. The tRNAs are differentially expressed; tRNA al" is transcribed in the opposite direction to U6, whereas tRNA It~ is not transcribed. Possible base-pairing between U6 and the U2 and SL RNAs, similar to the interactions that take place in the nematode trans-splicing system, are proposed.
In trypanosomatids all mRNA contain a 39-nt spliced leader (SL) at the 5' end. The SL sequence is acquired from a small RNA, the SL RNA, by trans-splicing. Trans-splicing is analogous to cis-splicing, as it proceeds through two transesterification steps creating a Y-structure intermediate, analogous to the lariat of the cis-splicing reaction (Agabian, 1990). Trans-splicing, like cis-splicing, takes place on a RNP particle, which has
been shown to contain U snRNPs and the unique particle, SL RNP (Michaeli et al., 1990; Cross et al., 1991). U2, U4 and U6 have been shown to actively participate in trans-splicing (Tschudi and Ullu, 1990). Although the trypanosome U snRNAs are related in sequence to the cis-splicing homologues, they possess several unique features (Mottram et al., 1989). None of these small RNAs contain the Sm-binding site or bind Sm antigens (Michaeli et al., 1990; Palfi et al., 1991). Apparently, no U! or U5 exists in trypanosomes (Mottram et al., 1989),
Correspondence to: Dr. S. Michaeli, Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel. Tel. (972-8) 343 626; Fax (972-8) 468 256; e-mail: [email protected] #We wish to dedicate this paper to the memory of Professor Alexander Kohn (known to his friends as Leshek), a pioneer in the development of live viral vaccines, founder of the Journal of lrreproducible Results and a leading figure in the Israeli scientific community. Leshek strove for honesty in science, but did so ~vith a great sense of humor. We will all miss him.
Abbreviations: AMT, aminomethyltrioxsalen; bp, base pair(s); C., Crithidia; Cf C. fasciculata; kb, kilobases(s) or 1000 bp; L., Leptomonas; Lc, L. collosoma; Ls, L. seymouri; N, A or C or G or T; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PSE, proximal sequence element; R, A or G;; RNP, ribonucleoprotein; SDS, sodium dodecyl sulfate; SL, spliced leader; Sin, antigens against which a patient (Smith) with lupus syndrome made antibodies; snRNA, small nuclear RNA; SSC, 0.15 M NaC1/0.015 M Na~.citrate pH 7.6; T., Trypanosoma; Tb, T. brueei; TMG, trimethylguanosine; U6, U6 snRNA; U6, gene encoding U6; U snRNA, uridylic-acid-rich snRNA; UV, ultraviolet; Y, C or T.
INTRODUCTION
SSDI 0 3 7 8 - 1 1 1 9 ( 9 5 ) 0 0 0 4 8 - 8
140 and it has been suggested that the SL RNA may have a dual function serving both as a substrate as well as a Ul-like snRNA (Bruzik et al., 1988). Recently, a novel RNA termed spliced leader associated RNA (SLA) was identified in Trypanosoma brucei (Tb) and was proposed to be the trans-spliceosomal U5 homologue (Watkins et al., 1994). There are still many open questions regarding the mechanism of trans-splicing. For example, it is not known how the SL RNA interacts with the pre-mRNA during the trans-splicing reaction. The phenomenon of transsplicing has now been extended to other systems (Bonen, 1993), the nematodes being the most studied one (Nilsen, 1993). Recently, in a functional trans-splicing assay using aminomethyltrioxsalen (AMT)-dependent UV crosslinking, a direct interaction between the nematode SL RNA intron sequence and the U6 RNA was demonstrated (Hannon et al., 1992). The great interest in the U6 RNA originates from the many observations supporting the notion that U6 has a catalytic function in splicing (Wise, 1993). U6 is the most conserved spliceosomal U snRNA in both length and sequence (Brow and Guthrie, 1988). The hexanucleotide 5'-ACAGAG, located in the central domain of the RNA, is found in all U6 RNAs sequenced so far, and mutations in this region block the first or the second steps of the splicing reaction (Fabrizio and Abelson, 1990; Madhani and Guthrie, 1992; Datta and Weiner, 1993). Recent experiments provide evidence for an intimate association of the ACA sequence with the 5' splice site (KandelasLewis and S6raphin, 1993; Lesser and Guthrie, 1993), suggesting that the 5' splice site is delivered to the catalytic center by pairing with the U6 RNA. To identify potential features specific for trans-spliceosomal U6 RNA, we have cloned and sequenced the U6 gene from the monogenetic trypanosomatid Lc. Using this data and recent sequence data that became available from other trypanosomatids (Xu et al., 1994), we propose possible base-pairing between the SL and the U6 RNA, similar to the interaction that takes place in nematodes. To elucidate the regulatory elements that may control the transcription of the U6 gene, we have analyzed the upstream sequence and revealed two tRNA genes which are differentially expressed and may serve as extragenic promoter elements of the gene.
EXPERIMENTALAND DISCUSSION (a) G e n o m i c organization of the U6 gene-linkage to two t R N A genes
To determine the copy number of the Leptomonas collosoma (Lc) DNA was Southern analysis with the homologous probe (Fig. 1). A single hybridization band
Lc U6 gene, subjected to Tb plasmid was observed
upon digesting genomic DNA with either an enzyme of a 6-bp recognition site (PvulI) or with enzymes of a 4-bp recognition site (Sau3A and TaqI), suggesting that the U6 is a single-copy gene. The sequence of the U6 gene, including 471 nt upstream and 392 nt downstream derived from HpalI subclones, is presented in Fig. 2. Since two genes (for tRNA TM and tRNA TM)were found upstream to the Tb U6 gene (Mottram et al., 1991), we examined whether such an arrangement also exists for the Lc U6 locus. Indeed, two tRNA genes were found, a Gin and a tRNAtuAA~ lie t R NAtctm) which have a transcriptional configuration identical to that of the Tb tRNAs. The sequence of the tRNAs and their inferred secondary structure is presented in Fig. 3. The box A of tRNA Ile is 5'-TAGCTCAGTCGG, which deviates from the canonical T R R Y N N A G T G G sequence by an additional C, whereas box B, 5'-GTTCGACCCC, agrees well with the consensus G T T C R A N N C C sequence (Geiduschek and Tocchini-Valentini, 1988). Also the box A of tRNA aln (5'-TAGTGTAGCGG) deviates from the consensus, whereas box B is 100% conserved. Similar to Tb Mottram et al., 1991) the distance between the first tRNA and the U6 gene is longer (99 nt) compared to the dis-
,,< kb
~
31_946 ::i !i:iii! 4.3
i li
i
2.3 m 2.0 m
0.5 m
Fig. 1. Southern analysis of Lc U6 sequence. Lc (Nelson et al., 1984) was provided by Dr. Nina Agabian (University of California, San Francisco, CA, USA). Genomic Lc DNA (10 ~tg) was digested with PvuII, Sau3A and Taql, separated on a 1.0% agarose gel, and transferred to Nytran membrane. The membrane was hybridized with the random-labelled Tb U6 gene probe. The Tb probe was a pGEM-3 vector carrying a 342-nt HaellI fragmentfrom the U6 locus (Mottram et al., 1989). Hybridization was at 65°C in 5xSSC/0.1% SDS/5× Denhardt's solution/100~tg per ml salmon sperm DNA, and the probe was washed by 0.1 x SSC at 65°C. Molecular mass markers (given in kb) are ~. DNA digested with HindllI.
141 •
-471
°
.
.
°
°
G G A C G C T G G G T A A A A C A C CAC G A A A ~ C G G A A A G T G G G G T A G C T G G G G T T C C C G A G T T G
-411
G TCAAAGGGGCAAGACTTAAGTTCTTGTGC GCAACGTTCGTGGGTTCGAACCCCACCTCC
-351
AGCATAGTTTTC C CTTTCC GCCGACTGGACATGTACCATGAAAGTCGCAGCGGCTGCCGA
-291
AGCTCTCATAGCTCAGTC GGTTAGAGCGTGGGTCTAATAAG CCCAAGGTCACAGGTTCGA
-231
C C C C T G T T G G G A G C A C T T T T C CAATC G C A C G T T T T T T C T T T T C G C G A C G G G A G A A A A A G T
-171
CACTCCTACCTGGACTCGAAC CAGGGTTATCGGATTCAGAGTCCGAGG TGATAACCGCTA
-111 -61
C A C T A T A G G A G C G C A C G C C T G T G C T G G C G TGGGC G T A G T T G T A C T T G C T A A T T C A C A A T A +1 C T A A G A T A C G G A A A A C A C G T G T C A C T G C G A A C G T T T T T T C C T C T A A G A A G C G G A G CC C C T
+i0
T C G G G G G A .CA,T C C A C A A C C TG ,,GAACTTCAACAGAGAAGATTAGCAC TC T C C C T G C G C A A G
+70
GCTGATGTCAATCTTCGAGAGATATAG CTTTTCGCCACGCTTTCCTCAC GTGTTTTCCCT
-371 t g g g t t c g a a c c ' c c a c c ~ c c a g c a t a g t ttt cc'ct tt c c g c c g a c t g g a c a t g t a c c a t g Ile tRNA -311 a a a g t c gc agc g g c t g c c g a a G C T C T C A T A G C T C A G T C G G T T A G A G C G T G G G T C T A A T A A -251 G C C C A A G G T C A C A G G T T C G A C C C C T G T T G G G A G C A c t t t t c caat cgc acgt t t t t t c t t box B -191 t t c gc ga c g g g ag a a a a a g t CACTCCTACCT~C~ACTCO.AACCAGGGTTATCGGATTCAGA GI.tR~A box B -131 O T C C G A G G T G A T A A C C G C T A C A C T A T A G G A ~ C g c a c g c c t gt gct g g c g t g g g c g t agt t box A -71 gtacttgctaattcacaatactaagatacggaaaacacgtgtcactgcgaacgttttttc -II c t c t a a g a a g c
Gln tRNA
* 130
TCAG T G G C T T A G A A G A C T A G T T C A C C G G A C G A G A G A G A T G G A G A G A G A G A G A G A A G C T G C
+190
C G C A C G G C T T G T G T G C G G G T T C C C C C G C C C C C C A C C C T C A C TCC C A A G C T C A C C G CGCAC
+250
CGTTTTCGGCGCGGGGGAGGGAGGGGTGCGCACCGCCCGACGGCGCCCGCCGCGAGCCCT
*310
TCC CCC CAC G C C C C A C GCG GG GCAAAC C CAAC C C A G C A C G C G C A C A C CACCC C A G G G A G A
4-370 C C C C G C C A G G C C C C C T C T A T C T T
392
Fig. 2. Sequence of the Lc U6 gene. The U6 gene was cloned from a kEMBL3 library carrying 15-20-kb Sau3A genomic fragments (Goldring et al., 1994) that was screened with the U6 plasmid probe (Mottram et al., 1989). Two positive clones carrying overlapping genomic segments were further used to subclone the gene. Fragments generated from digesting the phages with either TaqI, HpaII or Sau3A were subcloned into M13 and pGEM-3 vectors at the AccI and BamHI sites, respectively. Sequence analysis was performed using the dideoxy chaintermination method (Sanger et al., 1977) using flanking vector primers or the following synthetic oligos. Oligo #5958 is coding for nt + 136 to + 150, 5'-GGCTTAGAAGACTAG; oligo #6451 is an anti-sense to the tRNA ~j" nt - 162 to - 143, 5'-CTGGACTCGAACCAGGGTTA; and oligo #6452 is sense to #6451. The position of the first nt of the U6 RNA-coding sequence is designated as + 1. The sequence of the U6 RNA (101 nt) is underlined. This sequence data have been submitted to the EMBL/GenBank database with accession No. X79014. tance between the t R N A s (50 nt). Recently, the U6 gene was cloned and sequenced from Leptomonas seymouri and Crrithidiafasciculata (Xu et al., 1994). Identical c o m p a n ion t R N A s were found upstream to these t r y p a n o s o m a t i d U6 genes. As previously p r o p o s e d on the basis of r R N A (Fernandes et al., 1993), the conservation in the U6 c o m p a n i o n t R N A also suggests that Crithidia and Leptomonas are closely related to each other m o r e than each of them to Tb. The distance between the the the first t R N A and the U6 gene is highly conserved (97, 97, 98 and 99 bp in Tb, C f Ls and Lc, respectively), suggesting that this spacing m a y have functional significance. To examine the expression of both the U6 and the c o m p a n ion tRNAs, the entire clone, as well as specific probes c o m p l e m e n t a r y to the U6 and the two tRNAs, were used in N o r t h e r n analysis. The results are presented in Fig. 4, and indicate that at least two R N A transcripts are encoded by the U6 locus. Since the difference between the two t R N A s are only 2 nt, the expression of the individual t R N A s was examined using specific probes for each of the tRNAs. A 285-nt NruI-HindIII fragment, specific for t R N A '~, and an oligo, c o m p l e m e n t a r y to the t R N A G~", were used. The results indicate that t R N A a~" is expressed (Fig. 4C-6) whereas the t R N A n~ is not (Fig. 4D-8). To examine the specificity of the probes used in the N o r t h e r n analysis, Tb R N A was used as a control.
D loop GcGAUGuGAA
G G U CG U A CG C G U A
A G C C G U A C G U G P loop C G CC ,~ A UGAcucGAU A U U G U C C GC cA C A G G U u CG G AGAGC U UU GUAA~G T ~ C loop GC ~ GC GO UG C A U A AAU t R N A II~
U GGAccUGA CCUGGuucG
~A
G U u A U C A C c U AU'" CG GC GC AU C A A UCUG anticodon
T~IJC loop
an~icodon
Fig. 3. The tRNAs upstream to the U6 gene-sequence and their deduced secondary structure. Homology to tRNA molecules was determined by comparing the sequence to sequences in the EMBL data Library, Nucleotide sequence Database, and GenBank release 70 from InteUiGenetics (Mountain View, CA, USA). Sequence analysis software package of the Genetics Computer Group version 7.0 from April 1991 by John R. Devereaux was used to determine the tRNAs structure. The inferred tRNA sequences are indicated by upper-case lettering. Consensus polIII promoter elements (box A and box B) are underlined. The direction of transcription is indicated with an arrow in each case. The predicted secondary-structure models of tRNAG~nand tRNAne are shown. The anticodon, TOC and D loops are indicated.
The hybridization data in Fig. 4A-l, B-3, C-5 and D-7 indicate that in contrast to the U6 gene, the primary sequence of the t R N A s are not very well conserved. Neither the Tb nor the Lc U6 upstream sequence contain elements that resemble the TATA box or the PSE elements found upstream to the m a m m a l i a n U6 gene ( L o b o and Hernandez, 1990). In addition, no internal or downstream box A and box B can be found in the sequence similar to the p r o m o t e r elements of yeast (Brow and Guthrie, 1990). The identification of the U6 prom o t e r awaits functional analysis. However, the close proximity of t R N A s to the U6 suggests that these genes m a y be functionally related, possibly by increasing the local concentration of R N A polymerase III and its specific transcription factors. Four changes were revealed by c o m p a r i n g the Lc to the Tb U6 R N A sequence. All the changes are located in either the 5' terminal loop or the central domain. Two nt additions in position 8 and 13 resulted in an increased 5' stem. Such an increased stem was also revealed in Ls and C f U 6 sequences (Xu et al., 1994). The other changes are A 2s of Tb to C 27 in Lc and A 32 in Tb to C 34 in Lc. In Ls the U27 corresponds to C in C f like in Le. The changes in this position lie within the sequence h o m o l o gous to the A A U U sequence found in almost all U6 RNAs. In Tb the h o m o l o g o u s sequence to A A U U is
142
A
B
I
nt
1
I
2
!
3
D
C
I
4
l !
I
5
6
7
l
8
217-201-190" 180" 160-147-123-ii0--
90-76-67--
Fig. 4. Northern analysis with the U6 and tRNA genes. RNA was extracted from cells using the LiCl-urea extraction procedure as was previously described (Murphy et al., 1986). Total RNA ( 10 gg) prepared from Tb and Lc were resolved on a 7 M urea-6% polyacrylamide gel and electroblotted onto a Nytran membrane. Hybridization with labelled oligo was performed at 37°C in 5x SSC/0.1% SDS 5 x Denhardt's solution/100 gg per ml salmon sperm DNA, and the probe was washed at 37°C with 2 x SSC/0.1% SDS. (A) The Lc U6 HpaII subclone; (B) anti-sense oligo complementary to Tb U6 sequence nt 28-46, 5'-TCTTCTCTGTTGAATTTCC; (C), anti-sense oligo complementary to tRNA ~l" (# 6451 ); (D), a 285-nt (NruI-HindlIl) fragment carrying the tRNA ae. RNA from Tb was in lanes 1, 3, 5, 7 and Lc RNA in lanes 2, 4, 6 and 8. Size markers (pBR322 digested with HpalI) are indicated.
AACU, in Lc and Cf the sequence is ACCU and in Ls AUCU. Interestingly, in the nematodes, mutations within the AAUU sequence resulted in branch formation between the splicing substrate and the U6 (Yu et al., 1993). The sequence upstream to the conserved hexanucleotide ACAGAG is in all trypanosomatids UUCA, as opposed to the concensus sequence [CRA. (where a dot indicates that two or more nt differences were found among the sequences, including deletion or insertion of a residue) or CRAY (after excluding the trypanosomatid U6 sequences from the list)I, found in other U6 RNAs (Guthrie and Patterson, 1988). The significance of these changes, if any, may be understood when more is known about U6 snRNA-pre mRNA interactions during the trans-splicing reaction. (b) The Lc U6 RNA and its potential to interact with the U2 RNA It is currently unknown what types of interactions take place during the trypanosome trans-splicing reaction that involve the U6 RNA. Work done by Watkins and Agabian (1991), demonstrated that the Tb 5' end of the U2 RNA can be cross-linked in vivo to the 3' end of the U6 molecule, anlogons to the U2-U6 interaction in the mammalian system. The cross-linked region was grossly
mapped to be downstream to nt 29 and 70 of the U2 and U6, respectively. In the mammalian system, a similar region (U2, 3-11; U6, 87-95), was proposed on the basis of psoralen cross-linking (Hausner et al., 1990) and by introducing mutations in this region that influence the efficiency of mRNA splicing (Wu and Manley, 1991). Interestingly, this area of interaction in yeast, is fairly tolerant to mutations (Madhani and Guthrie, 1992). A potential for base-pair interaction between the Lc U2 and U6 RNAs is presented in Fig. 5. The proposed interaction domain between the 5' end of the U2 and the 3' end of the U6 RNA is shorter (10 nt) in Lc compared to the structure previously proposed for Tb (15 nt) (Watkins and Agabian, 1991). The length of this interaction domain, therefore, resembles more the domain proposed for mammals (9 nt) (Wu and Manley, 1991) and nematodes (8 nt) (Yu et al., 1993). In addition, the model presented in Fig. 5 includes the possible conserved interaction domain of helix I a and b (Madhani and Guthrie, 1992). Mutations in this domain in yeast impaired both steps of splicing, suggesting that this domain may be involved in catalysis of both reactions (Madhani and Guthrie, 1992). Interestingly, this domain lies just upstream to the branch point interaction region (the GUAGUA box) which is missing from the trypanosomatid U2 RNAs (Mottram et al., 1989; Hartshorne and Agabian, 1990). The potential for simultaneous basepairing between the U2/U6 helix I and a U2/pre-mRNA may therefore be different in trypanosome trans-splicing compared to cis-splicing. (c) Putative SL-U6 interaction in trypanosomatids The recent evidence for interaction of the U6 RNA with the 5' splice site in mammalian and yeast cis-splicing systems, suggests that U6 RNA may replace the U1 RNA in base-pairing with the 5' splice-site region (Wise, 1993). Since in trypanosome trans-splicing the 5' splice site is always located on the SL RNA, one might expect to find a potential for base-pairing interaction between the 5' CGC Gu--AA C~G
cG C-G C-GC_ G
cc-~
U6
~, he]i~ I=~he%"k. U=A U~G
heli~ II
Fig. 5. Potential interactions of the Lc U2 and U6 RNA. The U2 sequence is from Hartshorne and Agabian (1990). The model includes the potential interaction in helix I a and b, according to (Madhani and Guthrie, 1992). The interaction in domain II is in agreement with the interaction domain suggested for mammals (Hausner et al., 1990) and nematodes (Yu et al., 1993) and is different from the structure proposed for Tb (Watkins and Agabian, 1991).
143 splice site region of the SL RNA and the conserved hexanucleotide ACAGAG. Indeed, inspection of all trypanosomatid SL RNA sequences indicate conservation of the UG sequence at position + 4 and + 5 that can pair with the first conserved nucleotides AC. Lc is the only trypanosomatid SL RNA in which SL RNA possesses the sequence UGU that can potentially interact with the entire ACA sequence. Interestingly, the Cf U6 RNA carries a C ~ T change at the second position of the ACAGAG sequence and a compensatory base change of the corresponding SL RNA at position + 5, supporting the notion that the 5' splice site/U6 RNA interaction may be shared between cis- and trans-splicing systems (Xu et al., 1994). The U6/5' splice site is believed to be one of the last interactions before catalysis. One may, however, expect to find interactions between these molecules that take place at earlier stages of the reaction. Such an interaction was indeed revealed in the nematode system between the 3' end of the U6 sequence and the region of the Sin-binding site in the SL RNA (Hannon et al., 1992). The SL/U6 interaction permits simultaneous basepairing of the U6 RNA with both the SL and U2 RNA, suggesting that the U6 and U2 may serve as a connecting bridge between the SL RNA and the pre-mRNA (Hannon et al., 1992). To examine whether such interactions may also take place in trypanosomes, we searched for potential base-pairing interactions between the U6 and the trypanosomatid SL RNA molecules. Such a putative interaction (presented in Fig. 6), was revealed by searching for a U6 RNA sequence complementary to the 'Sm-like' binding site, located between stem-loops II and III of the trypanosomatid SL RNA. The U6 sequence of all trypanosomatids tested is identical in the proposed interaction domain (nt 68-86 in Tb and nt 70-88 in the other trypanosomatids), whereas the SL RNA sequence in this region is different, except for the sequence U(3_41GGR, which is common to all trypanosomatid SL RNAs. The putative interaction domain between the Tb molecules is extensive and is composed of 16 nt, whereas in Leptomonas sp. and Crithidia only the first 6-8 nt have the potential to interact. In nematodes it has been demonstrated that despite the extensive potential for basepairing interaction of 19 nt between the SL and U6 RNA (shown in Fig. 6), only 11 nt most probably compose the interaction domain (Hannon et al., 1992). It has been long demonstrated that no Sm antigens exist in trypanosomes (Michaeli et al., 1990; Palfi et al., 1991) and that the trypanosomatid U snRNAs lack the Sm-binding site (Mottram et al., 1989). The existence of a canonical Sm-like site in the trypanosomatid SL RNA (Bruzik et al., 1988), especially in the Leptomonas and Leishmania sequences, was always puzzling. As elucidated from the nematode study, the Sm-binding site and adjacent four 3' nucleotides are sufficient to confer the SL RNA function (Harmon et al., 1992). The conservation of this
C) SL 3' O H
OH
~x~0 UOUAAACCUUC,C GAAGUG----~ U
Gppp~
6
n ema t ode
\ p~pX
I
113
G
O
13o S L
~,c?, ,7"~,,.~ ,.~9,7c.~,
3.
OH / AGAGCUUCUAACU~GA G U C ~ 6 Gppp
76
k
93
SL ~ GGACCGAGCUUUCGG III I • II,I U 6 AGAGCUUCUAACUGUAGUC 78 CC~
sT. i . ~ l . ~ . o~ ~. ?
,.,
o~
Tb pppX
LC
~.s
U6 AGAC-CU'UCUAACUGUAGUC 73 CG IGIAAGCC~ S L UUUUGAGG I.i.ll.i. " t" U6 AGAGCUUC U CUGU GUC AA A
cf
Fig. 6. Potential for SL and U6 RNA interaction in trans-splicing. The nematode interaction domain is from Hannon et al. (1992). The Sm-binding site of the nematode SL RNA is indicated. The Tb SL RNA and U6 sequences are form Milhausen et al. (1984) and M o n r a m et al. (1989), respectively. The Lc sequences are from (Milhausen et al. (1984) and Goldring et al. (1994). The Ls SL RNA is from Bellofatto et al. (1988) the Cf SL RNA sequence from Muhich et al. (1987). The U6 sequence of C f a n d Ls are are from Xu et al. (1994). The U6 sequence comprising the U6-SL interaction domain is nt 68-86 for Tb and nt 70-88 for all other trypanosomatid species.
domain in trypanosomes and nematodes may be dictated by its absolute requirement to interact with the U6 RNA for catalysis of the trans-splicing reaction. The validity of the proposed trypanosomatid SL-U6 interactions awaits corroborating biochemical evidence for SL-U6 crosslinking in this region. Conclusions (1) The Lc U6 RNA is essentially identical to the Tb sequence apart from four changes, all located in the 5' stem-loop and the central domain. (2) The upstream 5' flanking region of the U6 gene harbors sequences for two tRNA genes which are expressed differently, tRNA ~ln is found 99 nt upstream to the U6 gene and is transcribed in an opposite direction to that of U6 gene. In contrast, tRNA ~1°is located 45 nt upstream to tRNACln; it is encoded by the same strand as the U6 RNA, but it is not transcribed. These tRNA genes are most probably the extragenic elements controlling the transcription of the U6 gene. (3) A potential for base-pair interaction between the 5' end of the U2 RNA and the 3' end of the U6 RNA is proposed. The interaction domain includes helix Ia, Ib and II, which are analogous to the interaction domains in mammals and yeast. (4) Two types of interactions are proposed between the SL and the U6 RNA. One interaction (between positions +4 to +6 of the SL RNA intron (UGU) with the ACA
144 s e q u e n c e o f t h e U 6 h e x a n u c l e o t i d e ) , r e s e m b l e s the intera c t i o n o f the cis-splicing i n t r o n s e q u e n c e at t h e 5'-splicesite j u n c t i o n w i t h the s a m e c o n s e r v e d U 6 h e x a n u c l e o t i d e s e q u e n c e . T h e o t h e r is a trans-splicing-specific i n t e r a c t i o n t h a t i n v o l v e s t h e 3' e n d o f t h e U 6 R N A a n d the S L R N A at the ' S m - l i k e ' b i n d i n g site. T h i s i n t e r a c t i o n is b a s e d o n a c o n s e n s u s s e q u e n c e U t 3 _ a ) G G R , w h i c h is s h a r e d a m o n g t h e t r y p a n o s o m a t i d S L R N A a n d is s i m i l a r to the n e m a t o d e s e q u e n c e U~a)GGA, t h a t was p r e v i o u s l y s h o w n to i n t e r a c t in v i t r o w i t h t h e U 6 s e q u e n c e . ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m the J o h n D. a n d
Catherine
T. M a c A r t h u r
Foundation
to the
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