Recruitment of mRNA cleavage�polyadenylationmachinery by the yeast chromatinprotein Sin1p�Spt2pGitit Hershkovits, Haim Bangio*, Ronit Cohen, and Don J. Katcoff†

The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan 52900, Israel

Edited by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, and approved May 18, 2006 (received for review March 13, 2006)

The yeast chromatin protein Sin1p�Spt2p has long been studied,but the understanding of its function has remained elusive. Theprotein has sequence similarity to HMG1, specifically binds crossingDNA structures, and serves as a negative transcriptional regulatorof a small family of genes that are activated by the SWI�SNFchromatin-remodeling complex. Recently, it has been implicated inmaintaining the integrity of chromatin during transcription elon-gation. Here we present experiments whose results indicate thatSin1p�Spt2 is required for, and is directly involved in, the efficientrecruitment of the mRNA cleavage�polyadenylation complex. Thisconclusion is based on the following findings: Sin1p�Spt2 fre-quently binds specifically downstream of many ORFs but almostalways upstream of the first polyadenylation site. It directly inter-acts with Fir1p, a component of the cleavage�polyadenylationcomplex. Disruption of Sin1p�Spt2p results in foreshortenedpoly(A) tracts on mRNA. It is synthetically lethal with Cdc73p,which is involved in the recruitment of the complex. This reportshows that a chromatin component is involved in 3� end processingof RNA.

fir1 � HMG1 � poly(A) � PAF complex

S in1p is a yeast chromatin nonhistone protein that has beenshown to function as a negative transcriptional regulator of

several genes, including Suc2 (1), Ino1 (2), and SSA3 (3). Activityof the HO promoter, as measured from an HO promoter drivinga lacZ gene (4), is also affected by Sin1. Sin1 (also known as spt2in this context) mutants were identified as suppressors of Ty andinsertions in the 5� noncoding region of the HIS4 gene (5).Because the negative regulation of these genes is overcome bythe SW1�SNF chromatin-remodeling complex (4, 6, 7) and theC-terminal domain of Sin1p interacts with Swi1p (8), it wassuggested that a function of SIN1p is to somehow maintainchromatin compaction at specific loci in the chromatin. Petersonet al. (2) found a functional relationship between the C-terminaldomain (CTD) of RNA polymerase II and Sin1p, but these datawere not pursued further. Sequence analysis of Sin1p showedsequence similarity in two domains to HMG1 (6, 7), a knownchromatin protein. Work from our laboratory showed that Sin1pcan bind four-way junction and crossing DNA structures (9),supporting the idea that Sin1p binds DNA as it enters and exitsthe nucleosome.

In the context of a global mapping project, Tong et al. (10)reported that there is a synthetic lethal interaction between sin1and cdc73, a member of the PAF complex. The PAF complex,which accompanies RNA polymerase II during elongation, wasshown to have an important function in 3� end formation and inpolyadenylation (11–13). In addition, a functional interactionwas demonstrated between Sin1p and Hpr1p, which is associatedwith the PAF complex (14).

Most recently, evidence has been presented indicating thatSin1p�Spt2p plays roles in transcription elongation, chromatinstructure and genome stability (15). In that study, synthetic lethalinteractions were reported between sin1 and paf1, and betweensin1 and ctr9. In addition, growth defects were identified in sin1,

rtf1 double mutants. Paf1p, Ctr9p and Rtf1p are all members ofthe PAF complex. In the same study, using ChIP analysis, Spt2pwas found preferentially associated with DNA that is activelytranscribed, binding to 3� untranslated regions more frequentlythan expected. Indeed, careful examination of Sin1p-bindingsites on the DNA identified previously (16) shows that Sin1poften binds DNA downstream of ORFs (see Results).

All these data indicated that there might be a role for Sin1pin 3� end formation of mRNA in yeast. In this paper, we showthat Sin1p is associated with Fir1p, a component and positiveregulator of the RNA cleavage�polyadenylation complex (17).Furthermore, deletion of Sin1p, or mutations in its C terminus,result in foreshortened poly(A) stretches at the 3� termini ofmRNA. We present a model suggesting that the polyadenylationmachinery is recruited to the nascent RNA transcript by inter-actions with the heavily phosphorylated CTD of RNA polymer-ase II at serine-2 (18, 19), the PAF complex that accompanies thepolymerase (11–13), and Sin1p that is bound to the chromatinjust downstream of the ORF of many yeast genes.

ResultsSin1p Binds Downstream of Many Genes, Just Upstream of a MajorPolyadenylation Site. Sin1p is a chromatin component whosepresence affects the transcription of a number of genes as isdescribed above. Harbison et al. (16) mapped DNA-bindingtranscriptional regulators, including Sin1p (Spt2p), to the ge-nome by using a chromatin immunoprecipitation assay. Carefulexamination of their data indicates that Sin1p often bindsdownstream of ORFs. In many cases, the binding site is down-stream of two ORFs that are on opposing strands, precluding thepossibility that Sin1p is binding promoter sequences at thosepositions. We compared the locations of the Sin1p-binding siteson 12 different genes having high affinity to Sin1p to predicted(20) and actual polyadenylation sites. The actual polyadenylationsites were determined experimentally by using 3� RACE (seeMaterials and Methods). It is apparent from the results shown inFig. 1 that in almost all cases, Sin1p binds �50 to 100 bpupstream of the first polyadenylation site after the ORF. In somecases, the binding is a bit further downstream, but usually, in thecontext of chromatin, not more than one nucleosome away fromthe 3� end of the coding region. These data suggested that Sin1pmight play a role in 3� processing events of the nascent mRNAtranscript.

Sin1p Interacts Directly with Fir1p. We previously reported resultsfrom a two-hybrid screen (21, 22) by using Sin1p as bait. Inaddition to recovering the previously reported colonies contain-

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviation: CTD, C-terminal domain.

*Present address: Procognia Israel, Ashdod 77610, Israel.

†To whom correspondence should be addressed. E-mail: katcoff@mail.biu.ac.il.

© 2006 by The National Academy of Sciences of the USA

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Fig. 1. Schematic representation of Sin1p binding site and polyadenylation sites on selected genes. The last 500 nucleotides of the coding region plus 500nucleotides of the 3� noncoding region are displayed. The numbers on the abscissa are relative to the 3� end of the ORF. All genes selected scored with a highconfidence level for Sin1p binding (16). The thick green line denotes the ORF. The black vertical arrow denotes the Sin1p binding site. The black and blue graphsdenote predicted polyadenylation sites using the maximum-likelihood (ML) and the discrete state-space (DSM) models, respectively (20). The red arrows denoteactual polyadenylation sites determined by 3� RACE. Major polyadenylation sites are marked with a thick arrow, while more minor sites are marked with a thinarrow. A black horizontal arrow denotes the position of the left primer used in the 3� RACE. The gel at the right of each graph shows a sample 3� RACE lane foreach gene. Marker sizes are 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, and 50 bp.

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ing Cdc23 and Sap1, we also recovered colonies encoding aminoacids 178–876 of Fir1p in the same screen. A direct interactionbetween the two proteins was confirmed by showing that 35S-labeled, in vitro translated Fir1p (amino acids 173–876) boundGST-Sin1p fusion proteins derived from the N terminus of theprotein, whereas GST alone did not bind (Fig. 2). A foreshort-ened peptide containing amino acids 173–810 bound the GST-Sin1p equally well (data not shown). This data supports thepossible involvement of Sin1p in the 3� processing of nascentmRNA transcripts, because Fir1p was identified recently as apositive regulator of polyadenylation (17).

Several hypothetical functional domains in Sin1p have beendefined based on genetic, structural, and biochemical consider-ations (6, 9, 21, 23–25). These domains include two HMG1-likedomains, (amino acids 26–88 and 98–159), an acidic domain(amino acids 224–304), and a basic C-terminal domain (aminoacids 303–333). To address which of these domains interacts withFir1p, we synthesized portions of Sin1p fused to GST based onthe functional domains that have been suggested, bound them toglutathione-agarose beads, and asked whether the radiolabeledFir1p peptide 173–810 would bind to Sin1p fragments. As can beseen in Fig. 2, peptides containing the first HMG1-like domain(amino acids 41–99) bound the Fir1 peptide, whereas moleculescontaining the other domains did not bind Fir1p. A fusionpeptide containing only a part of this HMG1-like domain (aminoacids 1–64) was able to bind Fir1p, but less well than thosepeptides containing the entire domain. In previous work, weshowed that a Sin1p peptide containing this HMG1-like domaincould bind four-way junction DNA (9). Taken together, theseresults demonstrate that the first HMG1-like domain of theprotein can interact with both Fir1p and with DNA. Because itis unlikely that this peptide interacts with both DNA and Fir1psimultaneously, we suggest that interaction between Fir1p andSin1p probably results in Sin1p displacement from the DNA.

Lack of a Functional Sin1p Results in Foreshortened poly(A). To testwhether the interaction between the chromatin component(Sin1p) and the polyadenylation regulator (Fir1p) has functionalsignificance, we compared average poly(A) length in yeaststrains carrying deletions in one or both of these proteins. In ourassay, we isolated whole-cell RNA from each strain and labeledthe 3� termini with [32P]pCp by using RNA ligase. We thendigested all of the RNA in each sample except for the poly(A)by using an RNase T1�RNase A mix. Reaction products were

electrophoresed on sequencing gels and autoradiographed. Theresults can be seen in Fig. 3. Examination of lanes 1 and 2 clearlydemonstrate that Sin1 deletion results in a skewed distributionof poly(A) tails that are significantly shorter in the sin1 deletionthan in the wild type. These results indicate that Sin1p, achromatin protein, is required for normal polyadenylation.

Previous genetic and biochemical evidence (6, 9) showed thatthe C terminus of Sin1p has functional significance. We there-fore tested a strain lacking the nine terminal amino acids ofSin1p (Fig. 3, lane 3) in our polyadenylation assay. As can beseen, this strain gave intermediate poly(A) lengths.

Fir1 deletions are not lethal but result in foreshortenedpoly(A) tracts by 5–7 nt when compared with wild type in an invitro polyadenylation reaction by using cell extracts from those

Fig. 2. Association between Sin1p and Fir1p peptides in vitro. GST orGST�Sin1p bacterially produced fusion proteins were immobilized on 200-�lglutathione agarose beads. Fir1p peptides labeled with [35S]methionine wereproduced in the Promega TNT-coupled transcription-translation system. Tenmicroliters of the reaction was incubated with the glutathione agarose beads.After washing, bound proteins were eluted with glutathione, and three-quarters of the eluted volume was subjected to SDS�PAGE and autoradiog-raphy. The multiple bands represent partial Fir1p molecules that result frompremature transcriptional and translational termination, internal transcrip-tional initiation, and RNA and protein degradation. The arrow shows thefull-length protein.

Fig. 3. Poly(A) length determination of total cell RNA. End-labeled RNA wasdigested with RNase and loaded on a 15% polyacrylamide sequencing gel. Tenpercent of each reaction described in detail in Materials and Methods wasloaded per lane. After autoradiography, the phosphorimage was quantitatedby using IMAGEJ (see Figs. 6 and 7, which are published as supporting informa-tion on the PNAS web site).

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strains (17). Our in vivo results agree with those in vitro results,showing that fir1 deletions result in poly(A) tracts that aresomewhat foreshortened (Fig. 3, compare lanes 1 and 4). Strik-ingly, a strain carrying the sin1 deletion together with a fir1deletion had significantly shorter poly(A) tracts than the fir1deletion alone, suggesting that the Sin1p might contact addi-tional polyadenylation complex components in addition to Fir1p.

Neither Sin1p nor Fir1p Affect poly(A) Site Selection. Because ourresults intimated that Sin1p is involved in polyadenylation, weasked whether it or Fir1p are involved in selection of the site atwhich the mRNA is cleaved and polyadenylated. This questionwas addressed by performing 3� RACE on RNA samples thathad been isolated from wild-type, sin1 and fir1 strains, and a sin1,fir1 double mutant. Of the 21 reactions performed, four samplereactions are displayed in Fig. 4. No significant differences wereseen between the samples, indicating that neither Sin1p norFir1p participate in determination of the cleavage and polyad-enylation site.

DiscussionAlmost all mRNA transcripts in eukaryotes are polyadenylatedat their 3� ends by a complex of proteins that are largelyconserved throughout evolution. Polyadenylation has beenshown to be important for mRNA stability, its protection fromribonucleases, transcription termination, export from the nu-cleus, and translation (26). Polyadenylation occurs in conjunc-tion with transcriptional termination in several steps. Before orconcurrent with termination, the transcript is cleaved at specificsites, a tract of adenylate residues is added, and it is then trimmedin a transcript-specific manner. These reactions are highly co-ordinated and are carried out by a host of factors. In yeast, thesefactors include poly(A)-binding protein (Pab1p), cleavage andpolyadenylation factors CFIA, CFIB, CPF, and poly(A) nuclease

PAN (17). Each factor with the exception of CFIB is encoded bymultiple polypeptides. Additional regulatory factors are associ-ated with these complexes. These factors include Fir1p (876 aa)that positively regulates mRNA polyadenylation and Ref2p thatregulates it negatively.

Recent progress has been made that addresses how thecleavage�polyadenylation complex is recruited to the nascentRNA. The CTD of RNA polymerase II, which becomes pro-gressively more phosphorylated at serine-2 as it progressesduring elongation, interacts with Pcf11 (18, 19) and serves as atermination factor (27).

Sin1 is synthetically lethal with cdc73, paf1, and ctr9 (10, 15),all components of the PAF elongation complex. Recent dataindicate that the PAF complex facilitates the linkage of tran-scriptional and posttranscriptional events, particularly polyade-nylation (12). Although the PAF complex associates with RNApolymerase II during initiation and elongation, it is required forproper polyadenylation of at least a subset of transcripts (13). Inthe absence of Cdc73p, the remaining PAF components are notassociated with the transcribing polymerase, and polyadenyla-tion is foreshortened. It has been suggested that the PAFcomplex functions in 3� end formation by recruitment of the 3�end-processing factors (11). The synthetic lethality of Sin1 withcomponents of this complex, and the fact that cdc73 and sin1mutants individually result in foreshortened poly(A) tracts,imply that Sin1p and components of the PAF complex haverequired overlapping functions.

Nourani et al. (15) have shown that Sin1p is frequentlyassociated with the coding region of actively transcribed genesand that its presence is important for the maintenance of histoneH3 at transcribed regions. In addition, sin1� mutants displayincreased intrachromosomal recombination in actively tran-scribed regions (15). These data suggested that the nonsequencespecific association of Sin1p with the transcribed DNA helpsmaintain the integrity of the chromatin during transcription.

We propose that as the transcription complex approaches apotential cleavage�polyadenylation site, the cleavage�polyadenylation complex is recruited by three components, allassociated with the RNA polymerase II (Fig. 5): (i) The CTD ofRNA polymerase II, which becomes increasingly phosphory-lated at serine-2 (18, 28), interacts with Pcf11 in the CFIAcomponent of the polyadenylation complex (19). (ii) Members ofthe PAF complex that are associated with the RNA polymeraseII interact with as-yet-undetermined members of the polyade-nylation complex (suggested by ref. 13). (iii) Sin1p, which mayaccompany the elongation complex (15), specifically binds aDNA sequence in the 3� noncoding region, near the cleavage�polyadenylation site, and interacts with Fir1p in the cleavage�polyadenylation complex. If any of these three interactions isablated (by gene disruption), polyadenylation is less efficient,and average cellular poly(A) is shorter than in the wild type. Ifany two of the ‘‘recruiters’’ are missing, polyadenylation iscurtailed, and the result is lethality. This idea is supported byreported synthetic lethalities: (i) between ctk1, whose geneproduct phosphorylates the CTD at serine-2, and any of the PAFcomplex components (11); and (ii) between cdc73, paf1, or ctr9,whose gene products are part of the PAF complex, and Sin1 (10).

Although our data indicate that Sin1p participates in therecruitment of the mRNA processing�polyadenylation machin-ery and ChIP analysis (16) shows that it preferentially bindsDNA just upstream of the polyadenylation site, Sin1p is notrequired to identify the polyadenylation site.

Hpr1p, which appears to be involved in chromatin remodeling,has been shown to be associated with the PAF complex (14). At37°C, a strain lacking Hpr1p and expressing a semidominantsin1-2 allele (29) is unable to grow, whereas strains with a sin1deletion grow almost normally. It was suggested in that paperthat in the wild type, Hpr1p might function to facilitate the

Fig. 4. Sin1p does not affect poly(A) site selection. 3� RACE was performedon total RNA samples isolated from wild-type and mutant yeast. Relevantgenotypes are YIF3, fir1�; YIF9, sin1�, fir1�; SLL5, wild type; SLL7, sin1�;SLL101, sin1-324 (missing the C-terminal amino acids). Primers are listed inTable 1, which is published as supporting information on the PNAS web site.

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removal of Sin1p from the chromatin and that in its absence, inthe sin1-2 strain, sin1-2p may be bound to the chromatin moretightly, causing toxicity. That interpretation is consistent withanother observation that overexpression of the C-terminal ofSin1p (where the sin1-2 mutation is found) is toxic (8), but thatthis toxicity is suppressed in spt4, spt5, and spt6 strains. Each ofthese proteins, like Hpr1p, has been implicated in modificationof chromatin structure. The Spt4�Spt5 complex interacts genet-ically and physically with Spt6, presumably to mediate transcrip-tion through chromatin (30, 31). Interestingly, recent data haveshown a functional connection between Spt2p and Spt6p (15),and spt6 mutations can alter mRNA 3� end formation at least inone documented case (32).

Sin1p has been isolated in a protein complex that includesPub1p, a poly(A)� RNA-binding protein, Npl3, a poly(A)�

RNA-transport protein, TIF4631 and TIF4632, proteins thatassociate with the poly(A)-binding protein Pab1p, and Sgn1p,another RNA-binding protein (33). These data also supportSin1p involvement in RNA processing.

We propose that, concomitant with its recognition by thepolyadenylation complex, Sin1p is removed from the chromatin,allowing the transcription apparatus to pass with the recruited 3�processing machinery in tow. The notion that Sin1p is removedfrom the chromatin is supported by the fact that the same shortdomain of Sin1p binds both DNA and Fir1p, and it is unlikelythat both can interact with Sin1p simultaneously.

Sin1 initially was discovered as a suppressor of mutations inthe SWI�SNF complex (2, 7). Later, Perez-Martin and Johnson(8) showed that the C terminus of Sin1p physically associateswith components of the SWI�SNF complex and that this inter-action is blocked in the full-length Sin1p protein by the N-terminal half of the protein. Recently, it has been reported thatSWI�SNF can stimulate transcription even after promoter clear-ance (34) during elongation. Pollard and Peterson (1) reportedthat members of the ADA�GCN5 complex that can acetylate

both histones and Sin1p are synthetically lethal with swi1, acomponent of the SWI�SNF complex. Mutations in Sin1 restoreviability. Taking all these data together, we propose that theSWI�SNF complex, the ADA�GCN5 complex (via acetylation ofSin1p), and Hpr1p each facilitate the removal of Sin1p from thechromatin at a site downstream of the ORF of many genes.Continued research is necessary to elucidate the precise role ofeach of these factors in mRNA transcriptional termination andprocessing. The removal of Sin1p from the chromatin might bea prerequisite for the passage of the transcription apparatus,without which there can be no further processing of the mRNA.

We show that a chromatin component is required to completemRNA processing, providing a link between chromatin structureand RNA processing. As details of the gene expression machin-ery are explored, it is becoming apparent that sometimes unex-pected functional relationships exist between the different com-ponents (35–38). Further research will be necessary to determinethe details of how the mRNA processing machinery is recruitedand activated.

Materials and MethodsYeast Strains. SLL5 (wild type with respect to Sin1), SLL7(sin1�), and SLL101 (sin1 1-324) were gifts of L. Lefebvre(University of British Columbia, Vancouver; ref. 6). YIF3 isfir1::HIS3, leu2�, trp1�, ura3-52, his3-�200, ade2-101, lys2-801.YIF9 is YIF3 with the addition of sin1::LEU2. Cy110 issin1::TRP1, leu2�, trp1�, ura3-52, his3-�200, ade2-101, lys2-801.

Two-Hybrid Screen and Verification of Protein Interactions. Thetwo-hybrid screen by using Sin1p as a bait was carried out as inShpungin et al. (22). A plasmid termed p82 was isolated from thelibraries and was found to encode amino acids 173–876 of Fir1p.Plasmids p178-876 and p178-746 were constructed by PCRamplification of the partial Fir1 coding region by using p82 astemplate and inserting them between the BamHI and PstI sites

Fig. 5. Model for polyadenylation complex recruitment�activation. The polyadenylation complex is recruited or activated in three parallel interactions denotedby arrows: (i) Hyperphosphorylated CTD on serine-2 contacts Pcf11p that is part of CFIA. (ii) The PAF complex contacts unknown members of the polyadenylationcomplex. (iii) Sin1p, specifically associated with the DNA just upstream of a polyadenylation site, contacts Fir1p that positively regulates polyadenylation. To allowthe transcription apparatus to proceed, Sin1p must be displaced from the DNA by Hpr1p, the SWI�SNF complex, and the ADA�GCN5 complex. Therecruited�activated polyadenylation complex cleaves the mRNA downstream of where the Sin1p was bound and polyadenylates the transcript. If a componentof any of the pathways is missing, polyadenylation is less efficient, and average poly(A) lengths are shorter. Blue denotes core factors required for pre-mRNA3� processing. Yellow denotes polyadenylation factors. Pink denotes poly(A)-trimming factors. Stippled shapes denote factors that displace Sin1p from the DNA.The striped bar denotes the ORF. The line after the ORF denotes the 3� noncoding region.

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of pBluescript (Stratagene). Coupled transcription-translationof [35S]methionine-labeled Fir1p peptides was accomplished byusing the Promega TNT system. Plasmids encoding the GST�Sin1p fusions were described in ref. 9. The in vitro-binding assaybetween the Sin1p and Fir1p peptides was carried out as inNovoseler et al. (9).

poly(A) Length Determination. The determination of poly(A)length for total cell RNA was done essentially as described in ref.39. Briefly, [32P]pCp was ligated to the 3� ends of 0.5 �g of RNAin a 30-�l reaction by using 12 units of T4 RNA ligase (Fer-mentas, Burlington, ON, Canada). After a 24-h incubation at4°C, 40 �g of yeast tRNA was added, and all but the poly(A) wasdigested by a mix of 80 units of RNase T1 and 32 �g of RNaseA for 2 h at 37°C in a final volume of 80 �l. Finally, the enzymes

were neutralized by proteinase K and phenol�chloroform ex-traction and ethanol precipitated. The samples then were run ona 15% polyacrylamide sequencing gel and autoradiographed.Quantitative evaluation of the autoradiogram was done byanalysis of phosphorimager images by using IMAGEJ.

3� RACE. 3� RACE was performed on total cell RNA by using theFirstChoice RLM-RACE kit (Ambion, Austin, TX) accordingto the instructions of the manufacturer. The PCR uses an RNAtemplate and amplifies a DNA fragment between a primerspecific to a gene (see list in Table 1) and the 5� end of thepoly(A) segment at the 3� end of the RNA molecule.

We thank L. Lefebvre for strains. This work was supported by internalLife Sciences Faculty funding at Bar Ilan University.

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