InteractiveFly: GeneBrief

Spt6: Biological Overview | References

Gene name - Spt6

Synonyms -

Cytological map position- 5E4-5E4

Function - transcription factor, mRNA processing

Keywords - Transcriptional accessory protein, chromatin remodeling, nucleosome assembly, mRNA processing

Symbol - Spt6

FlyBase ID: FBgn0028982

Genetic map position - X: 6,161,847..6,169,019 [+]

Classification - Tex (Transcriptional accessory protein), Ribosomal protein S1-like RNA-binding domain

Cellular location - nuclear

NCBI links: Precomputed BLAST | EntrezGene

Spt6 was identified in Saccharomyces cerevisiae as a transcription elongation factor which interacts with histone H3 and H4 (Bortvin, 1996). Spt6 functions in chromatin disassembly. During transcription, Spt6 allows RNA Pol II to pass through the DNA template and re-establishes chromatin structure after RNA pol II passage (Adkins, 2006; Hartzog, 1998; Kaplan, 2003; Saunders, 2003; Swanson, 1992; Kok, 2007 and references therein). Specifically, in yeast the histone H3-H4 chaperone Spt6 mediates nucleosome reassembly onto promoters during transcriptional repression. In the absence of Spt6-mediated nucleosome reassembly transcription is sustained (Adkins, 2006). Mammalian Spt6 has been shown to bind phosphorylated serine of RNA polymerase II C-terminal domain through a primitive SH2 domain and recruit mRNA processing, surveillance, and export factors to responsive genes, thus providing a link between transcription and mRNA splicing and export (Yoh, 2007).

In Drosophila Spt6 is implicated in processing pre-mRNAs. Spt6 is recruited rapidly and robustly to sites of active transcription. Spt6 co-purifies with the exosome, a complex of 3' to 5' exoribonucleases that is implicated in the processing of structural RNA and in the degradation of improperly processed pre-mRNA. Immunoprecipitation assays of Drosophila nuclear extracts show that the exosome also associates with the elongation factor dSpt5 and RNA polymerase II. In vivo, exosome subunits colocalize with dSpt6 at transcriptionally active loci on polytene chromosomes during normal development and are strongly recruited to heat-shock loci on gene induction. At higher resolution, chromatin immunoprecipitation analysis shows that the exosome is recruited to transcriptionally active units of heat-shock genes. These data provide a physical basis for the hypothesis that exosome-mediated pre-mRNA surveillance accompanies transcription elongation (Andrulis, 2002).

The production of full-length mRNA transcripts requires the coordinated effort of many factors, including the general elongation protein Spt6, which is essential and conserved. In yeast, Spt6 interacts genetically and biochemically with transcription elongation factors that regulate the processivity of RNA polymerase II (Hartzog, 1998). Spt6 may also modulate chromatin structure during transcription, through direct interactions with histone H3 (Bortvin, 1996). dSpt6 is recruited to sites of active transcription and colocalizes with the elongating form of Pol II (Kaplan, 2000; Andrulis, 2000). On the basis of these observations, it was considered that dSpt6 might associate functionally with the transcriptional elongation machinery (Andrulis, 2002).

A biochemical approach was taken to identify the factors with which dSpt6 associates. Full-length dSpt6 was cloned, tagged with an epitope and expressed in a stable Drosophila cell line. Nuclear extracts containing dSpt6-Flag-His6 (dSpt6FH) were prepared and subjected to Flag affinity chromatography followed by glycerol gradient sedimentation. Several polypeptides co-purified with the tagged dSpt6 and were identified unequivocally. Bands were excised from the gel and the proteins were identified by a combination of peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and mass spectrometric sequencing (Andrulis, 2002).

Nine bands were identified with approximate stoichiometry to dSpt6 that represent homologues of the subunits of the yeast and human exosome. An additional band corresponded to untagged dSpt6, consistent with the self-association of Spt6 previously deduced from analysis of Saccharomyces cerevisiae protein complexes. These observations sugested that only fractions of the Drosophila Spt6 and exosome are associated physically. Nonetheless, this physical association could withstand stringent purification conditions (Andrulis, 2002).

The exosome is a multisubunit complex of 3' --> 5' exoribonucleases that has both nuclear and cytoplasmic functions (Butler, 2002). Exosome function is evolutionarily conserved, because four of the human exosome genes can complement their cognate mutant yeast alleles. Originally identified in yeast as a complex involved in the processing of specific ribosomal RNAs, the nuclear exosome also has been shown to be critical for degrading unspliced pre-mRNAs (Bousquet-Antonelli, 2000). The yeast nuclear exosome also seems to survey pre-mRNA polyadenylation and mRNA release from transcription foci (Hilleren, 2001). These studies have led to the proposal that the yeast exosome contributes to a checkpoint that monitors proper pre-mRNA processing activities (Andrulis, 2002).

The Drosophila dSpt6-associated exosome is composed of at least nine exosome subunits, comparable to the yeast and human core exosome complexes, which have at least ten subunits. Comparative analysis of the Drosophila exosome subunits with the yeast and human exosome subunits showed a high degree of sequence conservation. Unexpectedly, dRrp40 was not annotated in the National Center for Biotechnology Institute (NCBI) nonredundant (NR) database, but was found by using MS/MS-derived peptide sequences in a BLASTP (basic local alignment search tool protein) search of the whole Drosophila genome. The open reading frame (ORF) surrounding the match shared high identity with human Rrp40 and so was named dRrp40 (Andrulis, 2002).

Notably, the Drosophila dSpt6-exosome complex that was purified lacked the Rrp43 and Rrp45 subunits found in the yeast core exosome complex. This might reflect differences in the functional and structural properties of the core and the dSpt6-associated exosome complexes. Although an Rrp45 homologue (dRrp45) is present in the Drosophila genome, an Rrp43 homologue is not. This suggests either that Drosophila has an unrecognizable functional homologue of Rrp43 that did not co-purify with the dSpt6-associated exosome or that Rrp43 does not represent a core exosome component. dRrp45 may be either substoichiometric or absent in the dSpt6-associated exosome complex. The high sequence identity of most subunits indicates, however, that the interactions and functions of the exosome are conserved throughout evolution (Andrulis, 2002).

To confirm that the polypeptides co-purifying with dSpt6 represent an exosome complex, antibodies were raised to recombinant proteins corresponding to five of the exosome subunits. Antibodies against dRrp6, dMtr3, dRrp4, dSki6 and dCsl4, but not the pre-immune sera, specifically recognized polypeptides of the expected relative molecular mass (Mr) in nuclear extracts. Using these antibodies, dRrp6, dMtr3 and dRrp4 were detected in the Flag immuno-resin eluate from the dSpt6FH extracts but not from control extracts. These interactions were insensitive to RNAse A and were therefore direct protein-protein interactions (Andrulis, 2002).

Because both the purified yeast and human core exosome complexes have exoribonucleolytic activity in vitro, whether this dSpt6-exosome complex has the same activity was determined. The Flag immuno-resin eluate containing this complex had 3' --> 5' exoribonucleolytic activity in vitro, whereas the eluate from the mock-transfected cells had negligible activity. This indicates that the exosome has the capacity to function as an exoribonuclease while in association with the transcription elongation factor dSpt6 (Andrulis, 2002).

The exosome interaction with dSpt6 could be detected directly in nuclear extracts using exosome-specific antibodies. Antibodies specific for dMtr3 and dRrp4 immunoprecipitated not only other exosome subunits, but also dSpt6. To examine whether the exosome associates with additional transcription elongation factors, immune complexes precipitated from whole-cell extracts of cells transfected with dRrp4-Flag-His6 (dRrp4FH) were analyzed. dRrp4FH co-immunoprecipitated roughly 5% of the exosome complex in extracts and 1% of dSpt6, consistent with the dSpt6FH data. About 1% of the related general transcription elongation factor dSpt5 and the large subunit of Pol II were detected. These results indicate that the exosome associates with an elongation complex in Drosophila extracts (Andrulis, 2002).

The exosome has been suggested to function at or near sites of transcription. To investigate this, immunofluorescence studies were carried on Drosophila polytene chromosomes with antibodies to exosome subunits. Antibodies against dSki6 stained polytene chromosomes at over 100 loci, including several loci that have high transcriptional activity during development. For example, the developmental puffs at 2B, 74E and 90B showed strong labelling. This immunofluorescence staining pattern, like that of many transcription factors, was not static but followed the patterns of gene activation during salivary gland development. The dSki6 and dSpt6 staining patterns showed considerable overlap, suggesting that these factors generally colocalize, and perhaps function together, on polytene chromosomes. dCsl4 and dSki6 also colocalize. These observations support the hypothesis that dSpt6 and the exosome interact on chromosomes at sites of active transcription, although it cannot be ruled out that other interactions contribute to this colocalization (Andrulis, 2002).

The exosome has been shown to survey heat-shock pre-mRNAs in yeast. To determine whether this surveillance might be occurring co-transcriptionally in Drosophila, tests were performed to see whether the exosome is recruited to heat-shock loci. Immunofluorescence analysis of dSki6 showed staining at the principal heat-shock loci 67B, 87A, 87C, 93D and 95D. In addition, dSki6 was recruited to a transgenic locus containing hsp70-lacZ. Antibodies specific for dCsl4 showed an immunofluorescence pattern identical to dSki6. Antibody staining at these endogenous and transgenic loci was coincident with dSpt6 staining. Notably, dSki6 showed a redistribution to heat-shock sites, with a concomitant decrease in staining at non-heat-shock loci. Thus, similar to the general transcription elongation factor dSpt6, the exosome seems to be recruited to and associated with induced genes (Andrulis, 2002).

The association of the exosome was examined at higher resolution on heat-shock genes using chromatin immunoprecipitation (ChIP). Formaldehyde-fixed chromatin was isolated from Drosophila Kc cells, and protein-DNA complexes were immunoprecipitated using antibodies to heat-shock factor (HSF), dSpt6 and subunits of the exosome. The distribution of these factors was measured along the hsp70 gene using primer sets that would detect co-immunoprecipitated promoter/5' and co-immunoprecipitated 3' fragments. HSF, the transcription factor that binds to the hsp70 promoter, immunoprecipitated only the 5' end of the gene. By contrast, dSpt6 showed roughly a sixfold recruitment to the body of the hsp70 gene on gene induction. Antibodies specific for the exosome proteins dRrp6, dRrp4 and dCsl4 immunoprecipitated more hsp70 DNA under heat-shock conditions. The amount of recruitment of exosomal subunits was increased, on average, fivefold on both the 5' and the 3' end of hsp70. These data are consistent with immunofluorescence analysis and suggest that the exosome is recruited to the whole body of hsp70 on induction of this gene (Andrulis, 2002).

ChIP assays on hsp26 were carried using the same antibodies. Similar amounts of recruitment were observed for HSF and roughly an eightfold increase in the recruitment of dSpt6. Quantitative analysis of the recruitment of exosome subunits to hsp26 showed, on average, an 11-fold increase on heat shock. dRrp4 clearly showed the greatest increase at the 3' end of hsp26 and hsp70 in material immunoprecipitated from heat-shocked cells. These ChIP data corroborate the immunofluorescence analysis and suggest that the exosome, like dSpt6, tracks with the transcription elongation complex (Andrulis, 2002).

Pol II associates with several factors for efficient pre-mRNA processing and elongation. Previous genetic and colocalization studies support a direct role for Spt6 in transcription elongation. The interaction and colocalization of dSpt6 and the exosome at transcriptionally active genes indicates that the machinery for transcription elongation and the machinery for pre-mRNA surveillance function together in vivo. It is proposed that the exosome exerts these surveillance functions co-transcriptionally, through an association with Spt6 and elongating Pol II. Together, these observations are consistent with the hypothesis that the exosome surveys and degrades inappropriately made or processed pre-mRNA molecules that may otherwise clog transcription elongation, RNA processing or export pathways (Andrulis, 2002).

Chromatin reassembly appears to be is facilitated by Spt6 stabilizing nucleosomes via interaction with H3 and H4 and by FACT maintaining a stable interaction with both the remodeled nucleosome and the displaced H2A·H2B dimer

RNA polymerase II (Pol II) transcription through nucleosomes is facilitated in vitro by the protein complex FACT (Facilitates Chromatin Transcription). This study shows that FACT is associated with actively transcribed Pol II genes on Drosophila polytene chromosomes. FACT displays kinetics of recruitment and of chromosome tracking in vivo similar to Pol II and elongation factors Spt5 and Spt6. Interestingly, FACT does not colocalize with Pol III-transcribed genes, which are known to undergo nucleosome transfer rather than disassembly in vitro. These observations are consistent with FACT being restricted to transcription that involves nucleosome disassembly mechanisms (Saunders, 2003).

Nucleosomes are inhibitory to transcription. One mechanism of inhibition is by blocking the path of the elongating RNA polymerase. In eukaryotes, three different RNA polymerases exist: Pol I, II, and III. Studies in vitro suggest that at least two polymerases, Pol III and II, have distinct mechanisms by which they transcribe through nucleosomes. Pol III is able to transcribe chromatin under physiological conditions in a purified system, whereas Pol II transcription requires nucleosome disassembly (Saunders, 2003).

The Drosophila heat shock gene hsp70 has promoter and leader regions that are free of nucleosomes, and a promoter-proximal, paused polymerase that prime it for rapid activation. Upon hsp70 gene induction by thermal stress, Pol II encounters nucleosomes downstream of +130. The machinery that enables Pol II to transcribe through these nucleosomes has yet to be characterized. The nucleosome remodeling complexes SWI/SNF and NURF do not appear to play a role during hsp70 transcription elongation. Prime candidates that remain include FACT (Facilitates Chromatin Transcription) and elongation factors Spt5 and Spt6. FACT is a complex that comprises Spt16 and SSRP1 (Orphanides, 1999) and facilitates Pol II elongation through chromatin in vitro (Orphanides, 1998). Spt6 has histone chaperone activity (Bortvin, 1996), and Spt5 and Spt6 show considerable colocalization with the elongating Pol II on Drosophila polytene chromosomes (Andrulis, 2000; Kaplan, 2000). Also Spt5, Spt6, and Spt16 have mutant phenotypes similar to those of histone genes (Saunders, 2003).

To examine whether FACT is positioned to regulate chromatin structure during transcription in vivo, comparative immunofluorescence analysis was performed on Drosophila polytene chromosomes. Immunofluorescence staining reveals the global distribution of FACT (Spt16) relative to the hyperphosphorylated, elongating Pol IIo. Antibodies against either FACT or Pol IIo label many sites on chromosomes prepared from unstressed, developing, third instar larvae, several of which correspond to major transcriptionally active loci (e.g., ecdysone puffs at 74E and 75B). Merging the FACT and Pol IIo images reveals a striking coincidence of FACT with Pol IIo labeled sites. FACT is also at the nucleolus, the site of Pol I transcription. In contrast, FACT is absent from the tandem cluster of 5S ribosomal RNA (rRNA) genes at 56F1-2 that are transcribed by Pol III and known to be strongly stained by antibodies to Pol III-specific transcription factors (Saunders, 2003).

Colocalization of FACT with many Pol II-transcribed genes suggests that FACT is intimately involved in Pol II transcription. This point is further strengthened by the observation that, after a 20-min heat shock, Pol IIo and FACT redistribute to and concentrate at heat shock loci. Pol II is recruited to heat shock loci within seconds, and the first Pol II molecule progresses through the gene in less than 2 min. If FACT facilitates Pol II transcription through nucleosomes at hsp70, then it must be recruited with corresponding rapidity. The fluorescence staining of FACT during a time course after heat shock was examined at the transgenic sites 9D and 61A, which contain just one copy of the hsp70 gene in a known orientation. The distribution was examined of the promoter-restricted heat shock transcription factor, HSF, relative to both FACT subunits, SSRP1 and Spt16, at 9D (the same results were obtained at 61A). HSF and FACT are strongly recruited to the transgenic loci within 2.5 min of heat shock. Even at this early time, FACT resolves from the promoter-associated HSF. The merged image shows a red edge of FACT staining emerging to the right of HSF, indicating that FACT localizes further downstream than HSF. Spt5 and Spt6, factors known to associate with the entire transcription unit, are also recruited within 2.5 min to these heat shock loci. In contrast to HSF, the staining of Spt6 and Spt5 completely overlaps with that of FACT. As activation continues, the chromatin at 9D decondenses further creating a chromosomal 'puff,' and the differential staining of HSF and FACT becomes more apparent, whereas Spt6 still completely overlaps with FACT, and Spt5 mostly overlaps with FACT. Thus, FACT, Spt6, and Spt5 are recruited rapidly to hsp70 upon heat shock, and they associate with the same decondensed regions of the puff (Saunders, 2003).

To examine at higher resolution the temporal and spatial distributions of Pol II, FACT, Spt6, and Spt5 on hsp70, cross-linking and chromatin immunoprecipitation (ChIP) experiments were performed at various times after an instantaneous heat shock. A short (1 min) cross-linking time provided snapshots of the process of transcription elongation. The rapid activation of hsp70 is evident from the detectable recruitment of Pol II to the leader region after a 75-s heat shock. FACT is rapidly recruited to and enriched in the first region of hsp70 that is packaged into nucleosomes at this early time. FACT associates with the 3' end of the gene after 150 s, the same time Pol II is first detected at the 3' region. After a 5-min heat shock, FACT is enriched in the open reading frame (ORF), and the level of FACT in the 3' region is at least eightfold higher than any level of FACT detected upstream (-154). This upstream region is constitutively nucleosome-free, and the level of FACT on it does not increase upon heat shock. These data are consistent with FACT and the Pol II elongation complex cotranslocating along hsp70, starting at the first nucleosome (Saunders, 2003).

Spt6, like FACT, is rapidly recruited to the leader and 5' regions of hsp70 and is first detectable at the 3' end 150 s after heat shock. The greater occupancy of Spt6 in the nucleosome-occupied coding region is consistent with Spt6 being involved in the modulation of the chromatin structure. Overall, the kinetic data place FACT and Spt6 at the correct time and place to contribute to cotranscriptional nucleosome disassembly on hsp70 in vivo. Although no reduction in hsp70 RNA was detected when FACT or Spt6 protein levels are depleted a few fold by RNA interference (RNAi) treatments of Kc cells, these proteins are abundant and may be in excess, especially during heat shock when the general reduction of transcription of most genes presumably increases the availability of elongation factors (Saunders, 2003).

Another factor implicated in the control of transcription through chromatin, Spt5, is also recruited to hsp70 upon heat shock and tracks along the gene with kinetics similar to Pol II, FACT, and Spt6. In contrast to FACT and Spt6, the level of Spt5 associated with hsp70 is less at the 3' end of the gene than at the leader region after a 5-min heat shock. Even before heat shock a strong signal for Spt5 can be detected in the region of the paused polymerase (leader region). Spt5 is known to have a role with the NELF complex in restricting Pol II's elongation early in the transcription cycle and in stimulating the mRNA 5'-capping machinery, activities that require its association with the leader region. Thus, in addition to a positive role in elongation, Spt5 appears to have a role that is both spatially and temporally separate from that of FACT and Spt6 (Saunders, 2003).

Coimmunoprecipitation in Drosophila nuclear extracts provides support for physical associations of Spt5, Spt6, FACT, and elongationally active Pol II. These results are consistent with those from yeast that support the idea that multiple Spt5 complexes exist, one of which is an elongation complex that includes Spt5, Spt6, and FACT (Lindstrom, 2003). Other elongation factors, the Paf1 complex and the chromodomain adenosine triphosphatase (ATPase), Chd1, also show physical and genetic interactions with FACT, indicating that transcription elongation through chromatin in vivo involves a sophisticated molecular machine (Saunders, 2003).

Nucleosome reassembly after transcription-induced disassembly is essential for the integrity of chromatin structure. A link has been established between Spt6 and nucleosome reassembly is known, and recent genetic evidence suggests a similar role for FACT. This study has demonstrated that upon hsp70 induction, FACT and Spt6 are strongly recruited to regions of hsp70 occupied by nucleosomes. Spt6 has been shown to interact with H3 and H4, and FACT with H2A and H2B. In the accompanying paper (Belotserkovskaya, 2003), it is shown that the SSRP1 subunit contacts the H3·H4 tetramer of the disassembled nucleosome, while Spt16 maintains interaction with the displaced H2A·H2B dimer. It is appealing to speculate that chromatin reassembly is facilitated by Spt6 stabilizing the nucleosomes via interaction with H3 and H4 and by FACT maintaining a stable interaction with both the remodeled nucleosome and the displaced H2A·H2B dimer. Whether disassembly or reassembly is the most critical function of FACT in vivo remains an intriguing question (Saunders, 2003).

Paf1 coordinates changes in nucleosome structure with transcription activation and Pol II elongation by recruiting Spt6

The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. The Drosophila homologs of three subunits of the yeast Paf1 complex have been identified and characterized and striking differences were found between the yeast and Drosophila complexes. Although Drosophila Paf1, Rtf1, and Cdc73 (Hyrax) colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure. Paf1 therefore directs the histone methyltransferase activity and links active transcription and modifications of chromatin structure. The data support a model in which the Drosophila Paf1 complex plays a key role in coordinating histone modifications and changes in nucleosome structure with transcription activation and Pol II elongation, thereby serving as a critical link between gene expression and chromatin structure (Adelman, 2006; full text of article).

Proper control of gene expression is necessary for the development, differentiation, and survival of the cell, and transcription regulation is a cornerstone of this process. The formation of mRNA in eukaryotes involves a complex multistep pathway wherein each step provides an opportunity for regulation. Once RNA polymerase II (Pol II) has been recruited to a promoter and initiates transcription, it must efficiently escape from the promoter-proximal region and transcribe through a gene that is covered with nucleosomes. The nascent RNA must also be capped, spliced, polyadenylated, and exported to the cytoplasm before it can serve as a template for protein translation. Recent evidence from many laboratories indicates that there is a dynamic interplay between the protein complexes that carry out mRNA transcription, processing, and export, such that the efficiency of one step can have significant consequences for other steps in the pathway. For this reason, many factors that are required for the production of functional, mature RNA and were initially thought to directly stimulate Pol II transcription elongation have since been shown to elicit their primary effects on cotranscriptional processing or RNA export. Thus, a major goal towards understanding the mechanisms of transcription regulation requires the identification of both the direct and indirect activities of the numerous factors implicated in RNA production (Adelman, 2006).

The yeast Paf1 complex is one example of a factor that has been linked to a number of transcription-related activities. Yeast Paf1 is a complex of at least five polypeptides (Paf1, Rtf1, Cdc73, Leo1, and Ctr9) that has been implicated in processes as divergent as transcription initiation and elongation, modification of histone tails, phosphorylation of the Pol II C-terminal domain (CTD), RNA processing, and export. Although yeast Paf1 was originally thought to be an alternate mediator based upon its direct interactions with Pol II, it has since been found to be recruited throughout the body of active genes and to associate with the elongation-competent form of Pol II. Additional roles for the Paf1 complex have been suggested by the association of Paf1 with several RNA processing and export factors, such as Ccr4, the major yeast deadenylase, and Hpr1, a component of the THO complex that is involved in the export of mRNAs (Adelman, 2006).

Components of the Paf1 complex are nonessential in yeast, but mutations in Paf1 subunits confer sensitivity to 6-azauracil and generate Spt phenotypes, which are generally thought to signify defects in transcription elongation. In vitro transcription assays with naked DNA templates suggested that Paf1 and Cdc73 might directly stimulate transcription elongation; however, it is not clear what effects Paf1 has on elongation rates in vivo. In Saccharomyces cerevisiae, deletion of Paf1 or Cdc73 did not alter the distribution of Pol II on an active gene but dramatically decreased the chromatin immunoprecipitation (ChIP) signal observed for serine 2-phosphorylated (Ser2-P) Pol II. Consistent with a Ser2 phosphorylation defect, recruitment of 3' cleavage and processing factors was impaired in the paf1Delta strain and poly(A) tail length was modestly shortened (Adelman, 2006).

A link between the Paf1 complex and the chromatin architecture within transcribed regions has been suggested by genetic interactions between Paf1 components and Chd1, subunits of the yeast FACT complex, and histone assembly factors in the Hir/Hpc pathway. The packaging of template DNA into nucleosomes is known to represent a formidable obstacle to Pol II elongation in vitro, an obstacle that is overcome in vivo by a number of proteins that facilitate Pol II elongation by modifying chromatin structure and/or stability. Examples of factors that have been implicated in transcription through nucleosomes are chromatin remodeling enzymes, such as Chd1 and Swi/Snf, and histone-binding proteins like Spt6 and FACT. The ensemble of these complexes appear to help disassemble nucleosomes to promote efficient Pol II transcription through bound DNA and then to reassemble nucleosomes after the passage of Pol II. Both Spt6 and FACT have recently been shown to help maintain the proper balance between assembly and disassembly of nucleosomes during active transcription by Pol II, with the loss of these factors leading to a net failure to reassemble nucleosomes in the wake of transcription (Adelman, 2006).

The yeast Paf1 complex is required for ubiquitination of histone H2B at lysine 123 in the promoter-proximal region of activated genes. This ubiquitination event is a prerequisite for the methylation of histone H3 (at lysine residues 4 and 79) that accompanies active transcription in yeast; thus, the latter processes are defective in cells lacking functional Paf1. In addition, the Paf1 complex has been reported to be critical for the recruitment of the yeast SET2 histone methyltransferase complex to actively transcribed genes, leading to methylation of histone H3 at residue lysine 36 (Adelman, 2006 and references therein).

Although the yeast Paf1 complex has been studied extensively, a number of important questions remain unanswered. Key questions concern the nature of the interactions between the subunits of the Paf1 complex and their associations with Pol II, as well as the importance of Pol II binding in Paf1 function. A pivotal issue concerns the fact that deletion of Rtf1 or Cdc73 has been reported to reduce the association of all Paf1 components with the Pol II and chromatin yet lead to much weaker phenotypes than does deletion of the other Paf1 components. These results have led some to propose that the critical role of Paf1 occurs when the complex is not chromatin associated; however, the other potential activities of Paf1 have yet to be clearly identified. Furthermore, the subunit composition of the Paf1 complex in human cells appears to differ from that in yeast, since the human Rtf1 protein does not appear to stably associate with the other members of the Paf1 complex (Adelman, 2006).

To address these issues and to investigate the activity of Paf1-associated proteins in Drosophila, the Drosophila homologs of the yeast Paf1, Rtf1, and Cdc73 proteins were identified and characterized. In vivo analyses of the Drosophila Paf1 complex uncover both important similarities to and differences from the reported functions of Paf1 in yeast and provide insight into the connections among histone methylation, nucleosome stability, and transcription activation in a metazoan organism. Strikingly, the Drosophila Paf1 homolog is a previously annotated gene that encodes an essential protein, suggesting that the role of Paf1 has evolved and become more critical in metazoans. Rtf1 is not stably associated with the Drosophila Paf1 and Cdc73 proteins in vivo and shows only a weak interaction with Pol II. Moreover, when Paf1-depleted cells are assayed by tandem RNA interference (RNAi)-ChIP, no changes were observed in the level of Ser2-P Pol II on the Hsp70 gene, in contrast to results obtained with yeast. Interestingly, it appears that major effects of Paf1 depletion are the loss of H3-K4 trimethylation near the Hsp70 promoter and a significant decrease in the recruitment of Spt6 and FACT to the body of the Hsp70 gene, suggesting that Drosophila Paf1 may coordinate the activities of elongating Pol II with factors that maintain the proper chromatin architecture during transcription (Adelman, 2006).

To survey for effects of Paf1 and Rtf1 depletion on the recruitment and distribution of Pol II and transcription factors at Hsp70, a series of ChIP assays were perfomred with RNAi-treated Drosophila cells. Analysis of Pol II distribution at Hsp70 in cross-linked material from LacZ or Paf1 RNAi-treated cells (10-min heat shock) showed no significant differences in either the distribution or the density of Pol II (Rpb3) as a result of Paf1 depletion. However, under these conditions, it was noted that the density of actively transcribing Pol II throughout the Hsp70 gene is extremely high (approximately one Pol II every 100 bp), so that it might be difficult to detect a modest decrease in the rate of transcription elongation (which would further increase Pol II density if the initiation rate remained unchanged). Although an approximately fivefold reduction was detected in the ChIP signal for Paf1 in the Paf1-depleted cells, no change was detected in the levels of transcription factor Spt5 or the Ser2-P form of Pol II in the Paf1-depleted samples. Thus, the reduced accumulation of Hsp70 RNA observed in Paf1-depleted cells does not appear to result from (1) a decrease in the overall recruitment of Pol II or transcription elongation factor Spt5 to the gene or (2) inefficient phosphorylation of Ser2 on the Pol II CTD (Adelman, 2006).

To confirm these results, similar ChIP experiments were performed with independent LacZ-treated or Paf1-depleted samples that were heat shocked for only 5 min before cross-linking. The data are in agreement with data demonstrating that depletion of Paf1 does not considerably alter the recruitment or progression of Ser2-P Pol II along the Hsp70 gene, nor does it affect the association of transcription elongation factor Spt5 (Adelman, 2006).

Because the distribution of Paf1 so closely mimicked that of Spt6 and the SSRP1 subunit of FACT during heat shock activation, whether depletion of Paf1 altered the recruitment of these factors to Hsp70 was investigated. Interestingly, significantly lower levels of both Spt6 and SSRP1 are observed at the Hsp70 gene in the absence of Paf1, suggesting that the association of Paf1 with elongating Pol II may facilitate the recruitment of Spt6 and FACT to a transcribed gene. To ensure that the decrease in the Spt6 and SSRP1 signals obtained at Hsp70 in ChIP assays was not due to a global depletion of these proteins, Western blot assays were performed that revealed that Spt6 and SSRP1 were present at normal levels in Paf1-depleted cells (Adelman, 2006).

To test for a role for Rtf1 in recruiting chromatin-associated factors to active genes, ChIP assays were also performed with Rtf1-depleted cells. These results reveal a modest decrease in the recruitment of Spt6 and SSRP1 to the active Hsp70 gene in the absence of Rtf1, while levels of Pol II and Spt5 remain largely unchanged. The more modest decreases in Spt6 and SSRP1 levels resulting from Rtf1 depletion are in agreement with the more subtle defects in Hsp70 transcription upon depletion of Rtf1 compared to depletion of Paf1 (Adelman, 2006).

These studies have provided new insights into the increased importance of the Paf1 complex in a metazoan system. It is significant that Paf1 is recruited in a manner that is spatially and temporally identical to that of chromatin-associated factors Spt6 and FACT. In agreement with the strong colocalization of Paf1 with these nucleosome-associated factors, it was shown that depletion of Paf1 significantly reduces the recruitment of both Spt6 and the FACT subunit SSRP1. A relationship among Paf1, Rtf1, and FACT is consistent with findings that an rtf1Delta mutation shows synthetic lethality with POB3, a subunit of the yeast FACT complex. Moreover, the FACT complex has been shown to interact with the Paf1 complex and the chromodomain-containing Chd1 protein at actively transcribed genes. In vitro, FACT has been shown to function optimally to facilitate transcription through nucleosomes when it is present at approximately one molecule of FACT per two nucleosomes; the effectiveness of FACT in promoting elongation is decreased dramatically below this threshold. If these results reflect the situation in vivo, the greater than 50% decrease in FACT levels at the active Hsp70 gene in Paf1-depleted cells would result in a rather pronounced effect on transcription through nucleosomes (Adelman, 2006).

Furthermore, recent evidence obtained with yeast has shown that mutations of Spt6 or the FACT subunit Spt16 lead to aberrant chromatin architecture in the wake of elongating Pol II, presumably due to defects in reassembly of nucleosome structure. The failure to efficiently repackage transcribed DNA results in transcription initiation from cryptic sites and a reduction in levels of properly initiated and processed RNA. If a primary role of Drosophila Paf1 is to help stably recruit factors like Spt6 and FACT, then loss of Paf1 activity could also lead to the accumulation of nonfunctional or improperly processed RNA species. In support of this idea, a paper that was published during the preparation of this report states that mutations in yeast Spt6 alter the recruitment of Paf1 subunit Ctr9 and lead to defects in 3'-end processing of nascent RNA. It is thus tempting to speculate that the vast array of transcription elongation and RNA processing and export defects reported in yeast Paf1 mutant strains could result from perturbation of the nucleosome structure along actively transcribed genes. Moreover, it may be these chromatin and processing defects that account for the decrease in the amount of Hsp70 mRNA that accumulates in response to heat shock in Paf1- or Rtf1-depleted cells (Adelman, 2006).

Finally, the Paf1 gene in yeast is nonessential while the Paf1 gene in Drosophila is essential. This may reflect the more varied and demanding requirements of the transcription machinery in higher eukaryotes, where chromatin frequently plays a greater and more stringent role in regulation. This, in turn, may place a greater demand on the Paf1 complex, which appears to function at the interface between transcription and chromatin, perhaps serving as a platform that stimulates the association of a number of nucleosome-modifying complexes with actively elongating Pol II (Adelman, 2006).

In summary, the gene for Paf1 is a required Drosophila gene that colocalizes with actively elongating Pol II when chromatin associated and plays a critical role in the activation of stress-induced genes. Furthermore, recent data reveal that mutations in parafibromin, the human homolog of the Paf1 complex subunit Cdc73, are associated with an elevated risk of parathyroid carcinomas; thus, the Paf1 complex may be a key regulator of cellular control in metazoans. The connection between Paf1 and trimethylation of histone H3 at lysine 4 near the promoters of active genes is particularly interesting because a human homolog of Trithorax, the histone methyltransferase implicated in this activity, is ALL-1/MLL-1, which is associated with a number of acute leukemias. Future work to define the way in which Paf1 directs the histone methyltransferase activity of this key enzyme should provide insight into the interaction between active transcription and modifications of chromatin structure. The data support a model in which the Drosophila Paf1 complex plays a key role in coordinating histone modifications and changes in nucleosome structure with transcription activation and Pol II elongation, thereby serving as a critical link between gene expression and chromatin structure (Adelman, 2006).

Drosophila histone locus bodies form by hierarchical recruitment of components

Nuclear bodies are protein- and RNA-containing structures that participate in a wide range of processes critical to genome function. Molecular self-organization is thought to drive nuclear body formation, but whether this occurs stochastically or via an ordered, hierarchical process is not fully understood. This question was addressed using RNAi and proteomic approaches in Drosophila to identify and characterize novel components of the histone locus body (HLB), a nuclear body involved in the expression of replication-dependent histone genes. The transcription elongation factor suppressor of Ty 6 (Spt6) and a homologue of mammalian nuclear protein of the ataxia telangiectasia-mutated locus that is encoded by the homeotic gene multisex combs (mxc) were identified as novel HLB components. By combining genetic manipulation in both cell culture and embryos with cytological observations of Mxc, Spt6, and the known HLB components, FLICE-associated huge protein (FLASH), Mute, U7 small nuclear ribonucleoprotein, and MPM-2 phosphoepitope, sequential recruitment and hierarchical dependency were demonstrated for localization of factors to HLBs during development, suggesting that ordered assembly can play a role in nuclear body formation (White, 2011).

Determining the mechanisms of nuclear body assembly and function is important for understanding how genomes express and maintain genetic information. This study idetified a total of 35 factors that when depleted by RNAi either disrupt HLB assembly, histone pre-mRNA processing, or both. Using several cytological and genetic assays, it is concluded that (1) essential components of Drosophila HLBs form bodies independently of histone gene expression, and (2) Drosophila HLBs form by hierarchical recruitment of components (White, 2011).

Nuclear bodies are categorized as activity independent or activity dependent. In many cases, transcription and processing of RNAs are the associated activities, suggesting that nuclear body formation can be coupled to gene expression. What is striking about Drosophila HLBs is their persistence in cells that are not replicating and, thus, not expressing histone genes. At least four components, Mxc, Mute, FLASH, and U7 snRNP, are present in HLBs in Drosophila embryonic cells that have exited the cell cycle. Similar conclusion have been reached for U7 snRNP by examining HLBs in postembryonic cells. Similarly, mammalian HLBs associated with the major histone gene cluster persist in serum-starved cells as judged by NPAT foci (White, 2011).

In contrast, the data show that Spt6 localizes to HLBs only when histone genes are transcribed. Consequently, by this criterion, HLBs would be considered activity dependent. Recruitment to HLBs of Spt6 and possibly other members of the transcription or processing machinery might reflect local accumulation at sites of high transcriptional activity, which occurs at the heat shock locus in response to a heat shock stimulus. Spt6 precipitates with MPM-2 in a phosphorylation-dependent manner, suggesting that another mechanism for Spt6 HLB localization might involve a specific interaction with a phosphorylated form of Mxc or another HLB protein (White, 2011).

Consistent with the notion that Drosophila HLBs are not strictly activity dependent, Mxc and FLASH first colocalize into foci during syncytial nuclear cycle 10, one cycle before zygotic histone transcription begins. Their association might be required for subsequent activation of histone gene expression. Mxc and FLASH also persist as colocalizing foci through all stages of mitosis, a time in the cell cycle when transcription is terminated and nascent transcripts are aborted. This observation differs from previous studies in mammalian cells that report the disappearance of NPAT foci during mitosis (White, 2011).

The prevailing model of nuclear body biogenesis invokes a process of self-organization of components, but whether such self-organization is entirely stochastic or involves a hierarchical relationship among components is a matter of debate. The current data suggest that aspects of both mechanisms participate in Drosophila HLB formation. If stochastic self-organization of individual components is sufficient for body formation, the absence of any one component should not affect the assembly of any other component into a body. If hierarchical self-organization plays a role in body formation, the localization of some factors will depend on the presence of others. It has been previously shown that loss of the variant histone H2aV prevents formation of Lsm11 foci but not of MPM-2 foci. This study shows that depletion of Mxc or FLASH prevents Mute and Lsm11 from forming nuclear foci. These genetic data suggest that stochastic self-organization alone is an incomplete description of Drosophila HLB formation and that hierarchical self-organization plays a role (White, 2011).

In the early embryo, Mxc/FLASH nuclear foci appear during cycle 10, whereas Mxc/FLASH/U7 snRNP/Mute/MPM-2 foci appear in cycle 11. It cannot be definitively concluded that the foci in cycle 10 give rise to the foci in cycle 11. Thus, one interpretation of these observations is that two different nuclear bodies are forming, possibly by different mechanisms. However, given the correlation to the timing of the onset of zygotic histone transcription in cycle 11, an interpretation is considered most parsimonious in which the formation of Mxc/FLASH foci represents an early step in HLB assembly that is necessary for subsequent recruitment of other HLB components. The persistence of Mxc/FLASH foci during mitosis while Spt6, Mute, Lsm11, and MPM-2 foci disappear is consistent with this interpretation (White, 2011).

It is therefore proposed that Drosophila HLB formation involves hierarchical tiers of assembly. Mxc and FLASH form the foundation of this hierarchy, and their assembly into HLB foci is not affected by the removal of other components: Mxc and FLASH colocalize into HLBs after loss of Mute in S2 cells and in Lsm11 mutant flies (Burch, 2011). Some observations of mammalian HLBs fit this model. Coilin localization to HLBs is disrupted in the absence of NPAT. FLASH is required for formation of NPAT foci and vice versa, and in cells in which coilin colocalizes to HLBs with NPAT and FLASH, coilin knockdown does not disrupt HLBs. Similarly, Drosophila coilin is present in the HLB of some but not all cells, and coilin mutants do not disrupt HLB formation (Liu, 2006; Liu, 2009; White, 2011 and references therein).

By tethering individual components of CBs or HLBs to a specific chromosomal location in mammalian cells, data has been provided in support of the stochastic self-organization model of nuclear body formation. These results are not necessarily in conflict with the current results. First, the tethering strategy indicates what can happen, not what does happen normally. Perhaps HLB assembly is normally hierarchical and involves some order of assembly, but upon manipulation, the order of assembly can change because the properties of stochastic self-organization of individual components can stimulate body assembly from multiple starting points. MPM-2 and Lsm11 form nuclear foci in Drosophila in the absence of histone genes, and this might reflect self-organizing properties resulting from stochastic interactions. FLASH can bind itself, and this study found that the two forms of Mute interact. Thus, a model is proposed in which hierarchical aspects of HLB assembly are driven by the set of possible interactions of individual molecules in a given genotype (i.e., wild type vs. mutant/knockdown) and during particular times in development and cell cycle progression. In a recent study, the tethering of paraspeckle protein components to a chromosome did not result in full paraspeckle formation. Rather, active transcription of the Men epsilon/β noncoding RNA is proposed to 'seed' paraspeckle formation (Mao, 2011). Similarly, Mxc/FLASH may seed HLB formation. Mxc: a novel participant in HLB assembly and histone mRNA biosynthesis (White, 2011).

The data suggest that Mxc is the functional equivalent of human NPAT. Mxc likely participates in both histone gene transcription and pre-mRNA processing and could possibly function to coordinate these processes. The essential role described for Mxc in HLB assembly supports the idea that HLBs are important for histone mRNA synthesis (White, 2011).

One of the more provocative findings of this study is that the mxc locus encodes a key HLB protein. mxc was named for leg bristle duplications displayed by hypomorphic mutants and is considered a member of the polycomb group of regulators that repress homeotic gene expression during development. The ectopic expression of homeotic genes (e.g., Ubx) in mxc mutants may result from a failure to synthesize sufficient replication-dependent histones, which are needed to form repressive chromatin. The cell-autonomous defect in cell proliferation caused by mxc-null mutations is also consistent with a role for Mxc in histone expression. Hypomorphic mxc alleles also cause hyperplasia of certain blood cell types that is suppressed by loss of Toll pathway signaling. Such phenotypic pleiotropy suggests that Mxc may regulate genes other than those encoding histones. These issues and the mechanisms of HLB assembly will benefit from further analysis of Drosophila Mxc (White, 2011).

Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster

The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription. They have a positive role in transcription elongation in Saccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells. In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo. To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, Drosophila homologs of Spt5 and Spt6 were identified and their localization was characterized on Drosophila polytene chromosomes. Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo (Kaplan, 2000; full text of article).

High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation

Drosophila transcriptional elongation factor Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock. Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription. Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5' and 3' ends of heat shock genes. Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF. These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo (Andrulis, 2000; full text of article).


Search PubMed for articles about Drosophila Spt6

Adelman, K., et al. (2006). Drosophila Paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26(1): 250-60. PubMed ID: 16354696

Adkins, M. W. and Tyler, J. K. (2006). Transcriptional activators are dispensable for transcription in the absence of Spt6-mediated chromatin reassembly of promoter regions. Mol. Cell 21: 405-16. PubMed ID: 16455495

Andrulis, E. D., Guzman, E., Doring, P., Werner, J. and Lis, J. T. (2000). High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. Genes Dev. 14: 2635-2649. PubMed ID: 11040217

Belotserkovskaya, R., et al. (2003). FACT facilitates transcription-dependent nucleosome alteration. Science 301: 1090-3. PubMed ID: 12934006

Bortvin, A. and Winston, F. (1996). Evidence that Spt6p controls chromatin structure by a direct interaction with histones. Science 272: 1473-6. PubMed ID: 8633238

Bousquet-Antonelli, C., Presutti, C. and Tollervey, D. (2000). Identification of a regulated pathway for nuclear pre-mRNA turnover. Cell 102: 765-775. PubMed ID: 11030620

Burch, B. D., et al. (2011). The interaction between FLASH and Lsm11 is essential for histone pre-mRNA processing in vivo in Drosophila. RNA 17(6): 1132-47. PubMed ID: 21525146

Butler, J. S. (2002). The yin and yang of the exosome. Trends Cell Biol. 12: 90-96. PubMed ID: 11849973

Hartzog, G. A., Wada, T., Handa, H. and Winston, F. (1998). Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. Genes Dev. 12: 357-369. PubMed ID: 9450930

Hilleren, P., McCarthy, T., Rosbash, M., Parker, R. and Jensen, T. H. (2001). Quality control of mRNA 3'-end processing is linked to the nuclear exosome. Nature 413: 538-542. PubMed ID: 11586364

Kaplan, C. D., Morris, J. R., Wu, C. and Winston, F. (2000). Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster. Genes Dev. 14: 2623-2634. PubMed ID: 11040216

Kaplan, C. D., et al. (2003). Transcription elongation factors repress transcription initiation from cryptic sites. Science 301: 1096-9. PubMed ID: 12934008

Kok, F. O., et al. (2007). The role of the SPT6 chromatin remodeling factor in zebrafish embryogenesis. Dev. Biol. 307: 214-226. PubMed ID: 17570355

Lindstrom, D. L. et al. (2003). Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Mol. Cell. Biol. 23: 1368-78. PubMed ID: 12556496

Liu, J. L., et al. (2006). The Drosophila melanogaster Cajal body. J. Cell Biol. 172: 875-884. PubMed ID: 16533947

Liu, J. L., et al. (2009). Coilin is essential for Cajal body organization in Drosophila melanogaster. Mol. Biol. Cell. 20: 1661-1670. PubMed ID: 19158395

Mao, Y.S., et al. (2011). Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs. Nat. Cell Biol. 13: 95-101. PubMed ID: 21170033

Orphanides, G. LeRoy, G., Chang, C., Luse, D. S. and Reinberg, D. (1998). FACT, a factor that facilitates transcript elongation through nucleosomes. Cell 92: 105-16. PubMed ID: 9489704

Orphanides, G., Wu, W., Lane, W. S., Hampsey, M. and Reinberg, D. (1999). The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 400: 284-8. PubMed ID: 10421373

Saunders, A, et al. (2003). Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo. Science. 301: 1094-6. PubMed ID: 12934007

Swanson, M. S. and Winston, F. (1992). SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae. Genetics. 132: 325-36. PubMed ID: 1330823

White, A. E., et al. (2011). Drosophila histone locus bodies form by hierarchical recruitment of components. J. Cell Biol. 193(4): 677-94. PubMed ID: 21576393

Yoh, S. M., Cho, H., Pickle, L., Evans, R. M. and Jones, K. A. (2007). The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev. 21(2): 160-74. PubMed ID: 17234882

Biological Overview

date revised: 15 December 2011

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