Molecular evidence for a positive role of Spt4 in transcription elongation

Rondón, Ana G.; García-Rubio, María L.; González-Barrera, Sergio; Aguilera, Andrés

doi:10.1093/emboj/cdg047

Cited by 88 publications

(105 citation statements)

References 56 publications

(92 reference statements)

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“…Spt4͞5 has been shown to act both positively and negatively on elongation by Pol II (8,9,11,13). The results of our ChIP analysis formally suggest that the processivity of Pol I is decreased Ϸ2-fold by the spt4⌬ mutation (Fig.…”

Section: Discussionsupporting

confidence: 69%

“…However, Spt4p and Spt5p also form a separate complex, devoid of Spt6p, that associates with Pol II physically and genetically, and this interaction is important for transcription elongation (8). More recent work has shown that deletion of the nonessential gene SPT4 results in reduced efficiency of Pol II elongation through GC-rich DNA sequences (9) and a general decrease in Pol II processivity (10). Taken together, all of these data clearly support a role for Spt4͞5 in transcription elongation by Pol II in yeast.…”

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confidence: 99%

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RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

Schneider

French

Osheim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

105

143

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Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30°C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.yeast Saccharomyces cerevisiae T he synthesis of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I is an important step in the synthesis of ribosomes, and its regulation is closely linked to the nutrient conditions and growth potential for the cell. Previous studies have identified essential components of the Pol I transcription apparatus as well as important cis-elements in Pol I promoters and revealed some potential mechanisms for regulation of transcription initiation (for reviews, see refs. 1-5). Unlike Pol II, however, little is known regarding mechanisms that regulate postinitiation steps of transcription by Pol I.One well characterized Pol II transcription elongation factor in yeast is a complex of two proteins, Spt4p and Spt5p (the complex will be referred to as Spt4͞5 here). The SPT4 and SPT5 genes were among many genes isolated for their ability to suppress transcription defects caused by insertions of the retrotransposon Ty1 (or the Ty1 long terminal repeats, ␦) in the 5Ј noncoding regions of yeast genes (6). Swanson and Winston (7) later showed that Spt4͞5 associates with Spt6p, which affects Pol II elongation through chromatin. However, Spt4p and Spt5p also form a separate complex, devoid of Spt6p, that associates with Pol II physically and genetically, and this interaction is important for transcription elongation (8). More recent work has shown that deletion of the nonessential gene SPT4 results in reduced efficiency of Pol II elongation through GC-rich DNA sequences (9) and a general decrease in Pol II processivity (10). Taken together, all of these data clearly support a role for Spt4͞5 in transcription elongation by Pol II in yeast.Spt4͞5 also plays a role in Pol II transcription elongation in mammalian cells. The mammalian homologues of Spt4p and Spt5p form a complex called the 5,6-dichloro-1-␤-Dribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF), which was originally identified as a factor that induces a DRB-dependent arrest of elongating Pol II complexes in reconstituted in vitro transcription assays (11). It was demonstrated that under nucleotide-limiting conditions, DSIF could also increase the rate of elongation of Pol II in vitro. Thus, work in mam...

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Section: Discussionsupporting

confidence: 69%

mentioning

confidence: 99%

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

Schneider

French

Osheim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

105

143

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“…Cells carrying mutations in SPT4 and SPT5 genes display phenotypes associated with defects in transcription elongation (39), and the gene products are thought to be involved directly in transcription elongation (30,32). However, the suppression of Rad26-independent TCR by Spt4-Spt5 does not seem to be achieved simply by stimulating transcription.…”

Section: Discussionmentioning

confidence: 99%

“…Immunoprecipitation studies showed that Spt4 and Spt5 form a complex, which physically interacts with Pol II (30). Yeast cells lacking Spt4 show reduced efficiency of Pol II elongation through GC-rich DNA sequences and a general decrease in Pol II processivity (31,32). These proteins are conserved eukaryotic transcription-elongation factors and are generally required for normal development and viral gene expression in multicellular eukaryotes (33).…”

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confidence: 99%

The C-terminal Repeat Domain of Spt5 Plays an Important Role in Suppression of Rad26-independent Transcription Coupled Repair

Ding

LeJeune

2010

Journal of Biological Chemistry

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In eukaryotic cells, transcription coupled nucleotide excision repair (TCR) is believed to be initiated by RNA polymerase II (Pol II) stalled at a lesion in the transcribed strand of a gene. Rad26, the yeast homolog of the human Cockayne syndrome group B (CSB) protein, plays an important role in TCR. Spt4, a transcription elongation factor that forms a complex with Spt5, has been shown to suppress TCR in rad26⌬ cells. Here we present evidence that Spt4 indirectly suppresses Rad26-independent TCR by protecting Spt5 from degradation and stabilizing the interaction of Spt5 with Pol II. We further found that the C-terminal repeat (CTR) domain of Spt5, which is dispensable for cell viability and is not involved in interactions with Spt4 and Pol II, plays an important role in the suppression. The Spt5 CTR is phosphorylated by the Bur kinase. Inactivation of the Bur kinase partially alleviates TCR in rad26⌬ cells. We propose that the Spt5 CTR suppresses Rad26-independent TCR by serving as a platform for assembly of a multiple protein suppressor complex that is associated with Pol II. Phosphorylation of the Spt5 CTR by the Bur kinase may facilitate the assembly of the suppressor complex. Nucleotide excision repair (NER)2 is a conserved DNA repair mechanism capable of removing a variety of helix-distorting lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs) (1). NER can be grouped into two pathways: global genomic repair (GGR), which refers to repair throughout the genome, and transcription coupled repair (TCR), which refers to a repair mechanism that is dedicated to the transcribed strand of actively transcribed genes (2). In the yeast Saccharomyces cerevisiae, Rad7, Rad16 (3), and Elc1 (4) are specifically required for GGR, but dispensable for TCR. Rad7 and Rad16 form a complex that binds specifically to UV-damaged DNA in an ATP-dependent manner and has DNA-dependent ATPase activity (5). Elc1 has been shown to be a component of a ubiquitin ligase that contains Rad7 and Rad16, and is responsible for regulating the levels of Rad4 protein in response to UV damage (6, 7). It has also been suggested that Elc1 is a component of another ubiquitin ligase complex, which contains Ela1, Cul3, and Roc1 but not Rad7 and Rad16 (8,9). The role of Elc1 in GGR may not be subsidiary to that of Rad7 and Rad16 (4).The mechanistic details of TCR are relatively well understood in Escherichia coli. The transcription repair coupling factor Mfd targets the transcribed strand for repair by recognizing a stalled RNA polymerase and actively recruiting the NER machinery to the transcription blocking lesion as it dissociates the stalled RNA polymerase (10). Conversely, the TCR mechanisms in eukaryotes appear to be extremely complicated (for reviews, see Refs. 11 and 12). In mammalian cells, Cockayne syndrome group A (CSA) and B (CSB) proteins are specifically required for TCR, but dispensable for GGR (13-16). Like its human homolog CSB, the yeast Rad26 plays an important role in TCR but is dispensable for GGR (17). Both human CSB (18) ...

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“…By contrast, Spt4 is a small polypeptide (100-120 amino acids) and is nonessential for cell growth in budding yeast (Malone et al 1993). Yeast spt4D cells display pleiotropic phenotypes that are probably linked to defects in transcription elongation and RNA processing (Rondó n et al 2003;Lindstrom et al 2003;Mason and Struhl 2005;Qui et al 2006;Schneider et al 2006). Many genetic interactions with spt4D have been observed in budding yeast (Saccharomyces Genome Database, www.yeastgenome.org).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

Schwer

Schneider²,

Pei³

et al. 2009

RNA

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The Spt5-Spt4 complex regulates early transcription elongation by RNA polymerase II and has an imputed role in pre-mRNA processing via its physical association with mRNA capping enzymes. Here we characterize the Schizosaccharomyces pombe core Spt5-Spt4 complex as a heterodimer and map a trypsin-resistant Spt4-binding domain within the Spt5 subunit. A genetic analysis of Spt4 in S. pombe revealed it to be inessential for growth at 25°C-30°C but critical at 37°C. These results echo the conditional spt4D growth phenotype in budding yeast, where we find that Saccharomyces cerevisiae and S. pombe Spt4 are functionally interchangeable. Complementation of S. cerevisiae spt4D and a two-hybrid assay for Spt4-Spt5 interaction provided a readout of the effects of 33 missense and truncation mutations on S. pombe Spt4 function in vivo, which were interpreted in light of the recent crystal structure of S. cerevisiae Spt4 fused to a fragment of Spt5. Our results highlight the importance of the Spt4 Zn 2+ -binding residues-Cys12, Cys15, Cys29, and Asp32-and of Ser57, a conserved constituent of the Spt4-Spt5 interface. The 990-amino acid S. pombe Spt5 protein has an exceptionally regular carboxyl-terminal domain (CTD) composed of 18 nonapeptide repeats. We find that as few as three nonamer repeats sufficed for S. pombe growth, but only when Spt4 was present. Synthetic lethality of the spt5 1-835 spt4D double mutant at 34°C suggests that interaction of Spt4 with the central domain of Spt5 overlaps functionally with the Spt5 CTD.

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Molecular evidence for a positive role of Spt4 in transcription elongation

Cited by 88 publications

References 56 publications

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

The C-terminal Repeat Domain of Spt5 Plays an Important Role in Suppression of Rad26-independent Transcription Coupled Repair

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

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