ASH1 mRNA localization in yeast involves multiple secondary structural elementsand Ash1 protein translation

Brief Communication

337

ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation Isabel Gonzalez, Sara B.C. Buonomo, Kim Nasmyth and Uwe von Ahsen Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cells’ differential mating-type switching during the subsequent cell cycle. This localization depends on actin filaments and a type V myosin (She1/Myo4). The 3′′ untranslated region (3′′ UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell’s bud [1,2]. Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA. We show here that, unlike all other known localization sequences that have been found in 3′′ UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region. A 77 nucleotide region stretching from 7 nucleotides 5′′ to 67 nucleotides 3′′ of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity. Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase. These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently. The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein. Address: Research Institute of Molecular Pathology, Dr. Bohrgasse 7, 1030 Vienna, Austria. Correspondence: Kim Nasmyth E-mail: [email protected] Received: 7 December 1998 Revised: 29 January 1999 Accepted: 2 February 1999 Published: 15 March 1999 Current Biology 1999, 9:337–340 http://biomednet.com/elecref/0960982200900337 © Elsevier Science Ltd ISSN 0960-9822

Results and discussion Secondary structure not primary sequence is important for the localizing activity of an element spanning the stop codon of ASH1

To identify the sequences responsible for the localizing activity of the ASH1 3′ UTR, we inserted sequences from it between GFP coding sequences and the CYC1 transcription terminator. These tripartite mRNAs were then expressed from the galactose inducible GAL1–10 promoter and their

cellular localization determined by in situ RNA hybridization using probes located within GFP (see Figure 1b). The endogenous ASH1 promoter is active only during anaphase and its replacement by the promoter from GAL1–10 permitted us to visualize mRNA localization at all stages of the cell cycle. To our surprise, the 3′ UTR of ASH1 (1,100 nucleotides 3′ of its stop codon) largely failed to direct GFP mRNA into buds (Figure 1a), but the inclusion of 200 nucleotides upstream of the stop codon permitted this to happen (data not shown). By removing fragments from the 5′ and 3′ ends, we identified a 77 nucleotide sequence (ASH1–U) that is sufficient to localize GFP mRNA to the bud tip; localization was dependent on the SHE1, SHE2 and SHE3 genes (Figure 1a and data not shown). This sequence stretched from 7 nucleotides before the stop codon to 67 nucleotides behind it. A 55 nucleotide insertion immediately behind the stop codon abolished bud localization (data not shown), implying that sequences 5′ and 3′ to the stop codon must be adjacent to each other. ASH1–U caused localization to bud tips in cells with small to medium buds, but it could not tightly anchor GFP mRNA to the distal cortex of buds during anaphase (Figure 1a). The secondary structure predicted for this region (Figure 1b) contains several double-stranded regions and is consistent with the pattern of adenine and cytosine sensitivity of in vitro transcribed RNA to dimethyl sulfate (data not shown). Each stem was mutated by exchanging the bases (marked by boxes, Figure 1b) from one strand with those on the opposing one, which should abolish base pairing in the region. All mutations that disrupted stem structures abolished the preferential mRNA accumulation in buds. In contrast, mutating seven out of nine bases in the terminal loop adjacent to stem III to the complementary sequence had no effect (data not shown). Restoration of stem I by compensatory base changes in the opposite strand restored mRNA localizing activity, as did restoration of stem III by exchanging the primary sequence (Figure 1c). Previous studies had not addressed the importance of the ten nucleotides from the coding sequences of ASH1 (needed for the formation of stem I). Our results imply that secondary structure rather than primary sequence (at least in the case of stems I and III) determines the recognition of this localizing element by factors that cause its transport into the bud. A double-stranded RNA-binding protein might therefore be involved [3–5]. ASH1 coding sequences required for mRNA localization

The localizing element in the neighborhood of the stop codon of ASH1 (ASH1–U) cannot alone be responsible for

338

Current Biology, Vol 9 No 6

correctly localizing endogenous ASH1 mRNAs. Tripartite mRNAs containing only this part of ASH1 accumulate in daughter cells, but they only partially localize at the cells’ distal cortex during anaphase. Furthermore, replacement of this region of ASH1 has little or no effect on the localization of mRNA [1]. To map localizing elements within the coding sequences of ASH1, we analyzed the distribution of various ASH1 deletion mutant mRNAs. Deletion of 800 nucleotides from the 5′ half (an interval flanked by two NsiI restriction enzyme sites; this deletion is designated Ash1∆N) or 1000 nucleotides from the 3′ half (the interval between the XbaI restriction site and the stop codon; this deletion is designated Ash1∆C) caused ASH1

(a) ASH1–U (S) ASH1–U (G2)

ASH1–U (anaphase)

ASH1–3′ UTR

she1∆ ASH1–U mRNA DAPI Nomarski

(b)

Fluorescent probes GAL1–10 GFP

mRNAs to accumulate throughout mother and bud cytoplasm (Figure 2a,b). Thus, neither the 5′-terminal half nor the 3′-terminal half is sufficient to localize mRNAs during anaphase in the context of the authentic ASH1 promoter. But, combination of the 5′-terminal half with 300 nucleotides from the 3′ region (Ash1∆C5) caused localization during anaphase, even in the absence of element ASH1–U (Figure 2a,b). Using the sensitive reporter mRNA localization system, both elements — the 300 nucleotides from the 3′ half (element C) as well as the region flanked by the NsiI sites, termed element N (Figure 2c) — independently directed GFP mRNA to small buds, in a SHE1-dependent manner, when expressed from the GAL1–10 promoter (Figure 2d). These data suggest that ASH1 contains at least three localizing elements, one in region N, one in region C, and a third spanning its stop codon and 3′ UTR (ASH1–U). No primary sequence similarity between the three elements was detected. The size of the elements N and C precludes any reliable RNA secondary structure prediction, making it impossible to compare them with the structure obtained for the ASH1–U element. Upon combining the three elements we found only a slight increase in efficiency of localization during anaphase; no difference between the various combinations (Figure 2d) or the whole open reading frame was found. Although the single element is sufficient for reporter mRNA localization, these results exclude a simple additive mechanism as an explanation for the requirement of the presence of elements N and C for authentic ASH1 mRNA localization.

CYC1t

Translation of the carboxy-terminal sequences of ASH1 is required for cortical anchoring ASH1 stop codon U 5′ A G A G A A 3′

UCUCUU

UG

A C

GC

U

Stem I

U UU C AA C U A CU CU A UA A

UCU C

Stem II

UGAUA CA A UGU

A

20

GA UA A

U A A UG UCUCUU

CUA UU

A C

C

U A A A A C A 60

A GA GA A

Stem III

A U C GU A U A GC A U

C A

U A A 40

A C U

ASH1–U

UCA A C U UA A

To address whether or not localization of ASH1 mRNA depends on its translation, we introduced a stop codon immediately after its AUG start codon (Figure 3). Western blotting confirmed that the resulting mRNA failed to produce any Ash1 protein, whereas northern blotting showed that it was as stable as wild-type ASH1 mRNA (data not shown). Untranslated ASH1 mRNA was less stringently localized than wild-type mRNA (Figure 3). It

(c) Figure 1

Reporter mRNA

Nomarski

Current Biology

(a) RNA in situ hybridization with GFP mRNA-specific probes. Localization of reporter mRNA to the daughter cell by the characterized cis-acting element ASH1–U at different cell-cycle stages and the failure of localization when using only the 3′ UTR or the ASH1–U element in a SHE1-deleted strain (she1∆). Upper panels show mRNA localization, middle panels show nuclei stained with DAPI and the lower panels show Nomarski light micrographs. (b) The reporter mRNA system and the predicted secondary structure of element ASH1–U based on thermodynamic considerations — from calculating the most stable structure. Sequences indicated with boxes were exchanged with those of the opposite strand and found to abolish localization. CYC1t , the CYC1 terminator. (c) Disruption of stem III leads to mislocalization (left) whereas primary-sequence-independent stem formation leads to accumulation of reporter mRNA in the bud (right).

Brief Communication

339

Figure 2 3' UTR ASH1 mRNA (c) GAL1–10 localization +

Coding sequence

(b)

ASH1 regions localizing GFP

+/– – – – –

Ash1∆N

Ash1∆N3

Ash1∆C

Ash1∆C5

DAPI

ASH1 mRNA

ASH1

– – –

+ – + + –

CYC1

GFP

ORF

ASH1 N

(d)

GAL–GFP ASH1 ORF

GAL–GFP ASH1–N

ASH1 3′ UTR C

GAL–GFP ASH1–C

GAL–GFP ASH1–N+U

U

GAL–GFP ASH1–C+U

GFP mRNA

Ash1∆N Ash1∆NU Ash1∆N1 Ash1∆N2 Ash1∆N3 Ash1∆N4 Ash1∆C Ash1∆C1 Ash1∆C2 Ash1∆C3 Ash1∆C4 Ash1∆C5 Ash1∆C6

TGA 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8

DAPI

ATG ASH1

Nomarski

(a)

Current Biology

(a) Schematic drawing of the ASH1 gene and various deletion constructs. Numbers in the ASH1 gene indicate 200 nucleotide intervals. All clones contain the authentic ASH1 promoter and a Myc9 epitope at the carboxy-terminal end of the Ash1 protein. A line in the 3′ UTR indicates the authentic ASH1 3′ UTR, a gray box indicates that the 3′ UTR was replaced by the one from Cdc6. A + indicates a similar extent of localization to that of the full-length ASH1 mRNA, +/–

indicates that a strong cytoplasmic background was observed. (b) Examples of RNA in situ hybridization with ASH1-specific fluorescent probes, either wild-type or with various deletions. (c) Overview of the cis-acting elements capable of directing GFP mRNA to the daughter cell. ORF, open reading frame. (d) Examples of in situ RNA hybridization experiments with elements or combinations of elements, as indicated, cloned behind the GFP gene.

was rarely associated exclusively with the distal cortex of the daughter cell and more frequently found throughout mother and daughter cell cytoplasm. Furthermore, unlike wild-type mRNA, it was frequently found at the bud neck in late anaphase cells. Similar results were obtained when we exchanged the ATG start codon for a TTG codon (data not shown). These results suggest that translation might be required for the anchorage of ASH1 mRNA at the distal daughter cell cortex.

mRNA. This suggests that Ash1 protein has a role in anchoring its mRNA at the distal cortex of daughter cells. Co-expression of a wild-type ASH1 gene did not suppress delocalization of the GFP–stop–ASH1 mRNA, a result which implies that Ash1 protein only promotes anchoring of mRNA from which it has been translated. A stop codon inserted in the middle of the coding sequences of ASH1 produced the same phenotype as that produced by one immediately following the AUG of ASH1, implicating the carboxy-terminal half of ASH1 in correct anchoring (Figure 3). An alternative explanation for the requirement for translation of the coding sequence of ASH1 in order for mRNA anchoring to occur, is unwinding of the mRNA by the ribosome which thereby unmasks a cis-acting element.

To test whether anchoring at the daughter cell cortex depends on the presence of Ash1 protein or translation of a construct containing the ASH1 coding sequence we compared the localization of a GFP–ASH1 fusion mRNA, translation of which produced a GFP–Ash1 fusion protein, with an otherwise identical mRNA differing only in having a stop codon between GFP and ASH1 coding sequences (GFP–stop–ASH1), translation of which produced GFP alone. Localization of the mRNA producing GFP–Ash1 protein resembled that of wild-type ASH1 mRNA, whereas localization of the GFP-producing mRNA resembled that of an untranslatable ASH1

Some aspects of the observed phenomena are similar to the requirement for Oskar protein in Drosophila development. It has been shown that the oskar gene product is important to keep the oskar mRNA anchored to the posterior pole of the Drosophila egg. Oskar protein can, however, act in trans, in contrast to our result with the Ash1 protein [6,7].

340

Current Biology, Vol 9 No 6

Yeast strains

Figure 3 Overall ASH1 ASH1 mRNA Bud-neck localization cytoplasmic partially mRNA localized anchored

All in situ RNA hybridization was performed in a diploid ASH1-deleted strain (K7219; ASH1:TRP1, otherwise W303 background). Strains carrying SHE gene disruptions were described previously [1].

Localization of ASH1 mRNA ASH1 mRNA

ASH1

73%

21%

0%

6%

In situ RNA hybridization was performed as described previously with CY3-fluorescent probes specific for either the ASH1 coding region [1] or GFP mRNA (four 50-mer DNA oligonucleotides used simultaneously). Indirect immunofluorescence microscopy was performed as described [13,14] with antibody 9E10 against the Myc epitope. Pictures were taken on a cooled charged couple device camera (Quantix, Photometrics) on a Zeiss Axioplan 2 microscope using the IP-Lab software package (Scanalytics, Inc.).

ASH1

20%

44%

14%

22%

Acknowledgements

24%

43%

15%

18%

We thank Ralf-Peter Jansen and Jürgen Knoblich for comments on the manuscript and Mark Petronczki for help with the ASH1–U mutagenesis.

ASH1

51%

42%

0%

7%

ASH1

21%

73%

18%

24%

24%

35%

17%

DAPI

Stop Stop ASH1

GFP

References

Stop GFP

+

ASH1

Stop GFP

ASH1

24% Current Biology

Comparison of ASH1 mRNA localization in cells containing untranslated or partially translated mRNAs, as identified by RNA in situ hybridization. All constructs contain the authentic ASH1 promoter.

We show that cis-acting elements required for mRNA localization are located within the coding region whereas in other mRNAs they have been found only in the 3′ UTR, with a single exception [8] (for a review see [9]). The three elements identified are independently sufficient to localize a reporter mRNA, and this redundancy of localizing elements has been observed only in rare cases [10,11]. Finally, only upon translation of the coding sequence of ASH1 can ASH1 mRNA become tightly associated with the distal cortex of the bud. This indicates either an involvement of the gene product in its mRNA localization or a ribosome-directed anchoring mechanism, or both.

Materials and methods Plasmids For the reporter mRNA system, the GFP coding sequence (about 700 nucleotides) including start and stop codons was cloned into the XbaI restriction site of expression vector p416Gal1 [12] and the various fragments from the ASH1 gene were cloned between GFP and the CYC1 terminator. Element ASH1–U and mutants were cloned as EcoRI–SalI oligonucleotides. Element N consists of an approximately 800 nucleotide fragment between the two NsiI restriction sites in the coding region. Element C covers nucleotides 1125–1445 (numbering starting at the first nucleotide of the start codon). For the deletion analysis, a SalI–SacI fragment of the ASH1 gene containing a Myc9 epitope at the carboxy-terminal end in Yeplac181 (c3565) was used. For the constructs indicated in Figure 2a, the ASH1 3′ UTR was replaced with that of CDC6 (c3551). For detection of Ash1 protein, the various deletions were cloned in vectors of the YCplac series. Stop codons behind the start codon were introduced by PCR using mutagenic primers and subsequent cloning using the adjacent SphI and NheI sites. All clones were confirmed by DNA sequencing.

1. Long RM, Singer RH, Meng X, Gonzalez I, Nasmyth K, Jansen R-P: Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 1997, 277:383-387. 2. Takizawa PA, Sil A, Swedlow JR, Herskowitz I, Vale RD: Actindependent localization of an RNA encoding a cell-fate determinant in yeast. Nature 1997, 389:90-93. 3. St Johnston D, Brown NH, Gall JG, Jantsch M: A conserved doublestranded RNA-binding domain. Proc Natl Acad Sci USA 1992, 89:10979-10983. 4. Finerty PJ, Bass BL: A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids. J Mol Biol 1997, 271:195-208. 5. Wilson SA, Brown EC, Kingsman AJ, Kingsman SM: TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of Flightless I. Nucleic Acids Res 1998, 26:3460-3467. 6. Markussen F-H, Michon A-M, Breitwieser W, Ephrussi A: Translational control of oskar generates Short OSK, the isoform that induces pole plasm assembly. Development 1995, 121:3723-3732. 7. Rongo C, Gavis ER, Lehmann R: Localization of oskar RNA regulates oskar translation and requires Oskar protein. Development 1995, 121:2737-2746. 8. Capri M, Santoni MJ, Thomas-Delaage M, Ait-Ahmed O: Implication of a 5′′ coding sequence in targeting maternal mRNA to the Drosophila oocyte. Mech Dev 1997, 68:91-100. 9. Bashirullah A, Cooperstocl RL, Lipshitz HD: RNA localization in development. Annu Rev Biochem 1998, 67:335-394. 10. MacDonald PM, Kerr K: Redundant RNA recognition events in bicoid mRNA localization. RNA 1998, 3:1413-1420. 11. Gautreau D, Cote CA, Mowry KL: Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocyte. Development 1997, 124:5013-5020. 12. Mumberg D, Muller R, Funk M: Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 1994, 22:5767-5769. 13. Jansen R-P, Dowzer C, Michaelis C, Galova M, Nasmyth K: Mother cell-specific HO expression in budding yeast depends on the unconventional myosin Myo4p and other cytoplasmic proteins. Cell 1996, 84:687-697. 14. Bobola N, Jansen R-P, Shin TH, Nasmyth K: Asymmetric accumulation of Ash1p in postanaphase nuclei depends on a myosin and restricts yeast mating-type switching to mother cells. Cell 1996, 84:699-709.