Spt4-Spt5 Complex Research Articles

The transient Spt4-Spt5 complex as an upstream regulator of non-coding RNAs during development.

The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline and somatic nuclei, massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.

The transcription factor Spt4-Spt5 complex regulates the expression of ATG8 and ATG41

ABSTRACT Macroautophagy/autophagy, a highly conserved dynamic process, is one of the major degradative pathways in cells. So far, over 40 autophagy-related (ATG) genes have been identified in Saccharomyces cerevisiae, most of which have homologs in more complex eukaryotes. Autophagy plays a crucial role in cell survival and maintenance, and its dysfunction is related to various diseases, indicating that the proper regulation of autophagy is important. Although the overall process of autophagy has been extensively studied, in particular with regard to the function of the Atg proteins, relatively little is known about the regulatory mechanisms that control autophagy activity. Spt5 is one of the transcriptional factors that is universally conserved across all domains. This protein can form a complex with Spt4, together playing a central role in transcription. In complex eukaryotic cells, the Spt4-Spt5 complex plays a dual role in gene regulation, acting both to delay transcription through promoter-proximal pausing, and to facilitate transcriptional elongation. In contrast, in S. cerevisiae, only the positive function of the Spt4-Spt5 complex has been identified. Here, we show for the first time that the Spt4-Spt5 transcription factor complex negatively regulates ATG genes in S. cerevisiae, inhibiting autophagy activity during active growth. Under autophagy-inducing conditions, the repression is released by Spt5 phosphorylation, allowing an upregulation of autophagy activity. Abbreviations AID: auxin-inducible degron; ATG: autophagy-related; ChIP: chromatin immunoprecipitation;Cvt: cytoplasm-to-vacuole targeting; DSIF: DRB sensitivity-inducible factor; NELF: negativeelongation factor; ORF: open reading frame; PA: protein A; PE: phosphatidylethanolamine;prApe1: precursor aminopeptidase I; RT-qPCR: real-time quantitative PCR; RNAP II: RNApolymerase II; TSS: transcription start site; WT: wild-type

TFS and Spt4/5 accelerate transcription through archaeal histone-based chromatin.

RNA polymerase must surmount translocation barriers for continued transcription. In Eukarya and most Archaea, DNA-bound histone proteins represent the most common and troublesome barrier to transcription elongation. Eukaryotes encode a plethora of chromatin-remodeling complexes, histone-modification enzymes and transcription elongation factors to aid transcription through nucleosomes, while archaea seemingly lack machinery to remodel/modify histone-based chromatin and thus must rely on elongation factors to accelerate transcription through chromatin-barriers. TFS (TFIIS in Eukarya) and the Spt4-Spt5 complex are universally encoded in archaeal genomes, and here we demonstrate that both elongation factors, via different mechanisms, can accelerate transcription through archaeal histone-based chromatin. Histone proteins in Thermococcus kodakarensis are sufficiently abundant to completely wrap all genomic DNA, resulting in a consistent protein barrier to transcription elongation. TFS-enhanced cleavage of RNAs in backtracked transcription complexes reactivates stalled RNAPs and dramatically accelerates transcription through histone-barriers, while Spt4-Spt5 changes to clamp-domain dynamics play a lesser-role in stabilizing transcription. Repeated attempts to delete TFS, Spt4 and Spt5 from the T. kodakarensis genome were not successful, and the essentiality of both conserved transcription elongation factors suggests that both conserved elongation factors play important roles in transcription regulation in vivo, including mechanisms to accelerate transcription through downstream protein barriers.

Diverse Roles of RNA Polymerase II-associated Factor 1 Complex in Different Subpathways of Nucleotide Excision Repair

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA polymerase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.

Core Structure of the Yeast Spt4-Spt5 Complex: A Conserved Module for Regulation of Transcription Elongation

The Spt4-Spt5 complex is an essential RNA polymerase II elongation factor found in all eukaryotes and important for gene regulation. We report here the crystal structure of Saccharomyces cerevisiae Spt4 bound to the NGN domain of Spt5. This structure reveals that Spt4-Spt5 binding is governed by an acid-dipole interaction between Spt5 and Spt4. Mutations that disrupt this interaction disrupt the complex. Residues forming this pivotal interaction are conserved in the archaeal homologs of Spt4 and Spt5, which we show also form a complex. Even though bacteria lack a Spt4 homolog, the NGN domains of Spt5 and its bacterial homologs are structurally similar. Spt4 is located at a position that may help to maintain the functional conformation of the following KOW domains in Spt5. This structural and evolutionary perspective of the Spt4-Spt5 complex and its homologs suggest that it is an ancient, core component of the transcription elongation machinery.

Analysis of a Splice Array Experiment Elucidates Roles of Chromatin Elongation Factor Spt4–5 in Splicing

Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4–5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4–5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4–Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.

Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins.

During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.

The Paf1 complex physically and functionally associates with transcription elongation factors in vivo.

We are using biochemical and genetic approaches to study Rtf1 and the Spt4-Spt5 complex, which independently have been implicated in transcription elongation by RNA polymerase II. Here, we report a remarkable convergence of these studies. First, we purified Rtf1 and its associated yeast proteins. Combining this approach with genetic analysis, we show that Rtf1 and Leo1, a protein of unknown function, are members of the RNA polymerase II-associated Paf1 complex. Further analysis revealed allele-specific genetic interactions between Paf1 complex members, Spt4-Spt5, and Spt16-Pob3, the yeast counterpart of the human elongation factor FACT. In addition, we independently isolated paf1 and leo1 mutations in an unbiased genetic screen for suppressors of a cold-sensitive spt5 mutation. These genetic interactions are supported by physical interactions between the Paf1 complex, Spt4-Spt5 and Spt16-Pob3. Finally, we found that defects in the Paf1 complex cause sensitivity to 6-azauracil and diminished PUR5 induction, properties frequently associated with impaired transcription elongation. Taken together, these data suggest that the Paf1 complex functions during the elongation phase of transcription in conjunction with Spt4-Spt5 and Spt16-Pob3.

Evidence that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-dependent transcription in vitro.

Recently, a positive and a negative elongation factor, implicated in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, has been identified. P-TEFb is a positive transcription elongation factor and the DRB-sensitive kinase that phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). PITALRE, a member of the Cdc2 family of protein kinases, is the catalytic subunit of P-TEFb. DSIF is a human homolog of the yeast Spt4-Spt5 complex and renders elongation of transcription sensitive to DRB. DRB sensitivity-inducing factor (DSIF) binds to RNA Pol II and may directly regulate elongation. Here we show a functional interaction between P-TEFb and DSIF. The reduction of P-TEFb activity induced by either DRB, antibody against PITALRE, or immunodepletion resulted in a negative effect of DSIF on transcription. DSIF acts at an early phase of elongation, and the prior action of P-TEFb makes transcription resistant to DSIF. The state of phosphorylation of CTD determines the DSIF-RNA Pol II interaction, and may provide a direct link between P-TEFb and DSIF. Taken together, this study reveals a molecular basis for DRB action and suggests that P-TEFb stimulates elongation by alleviating the negative action of DSIF.

Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae.

Previous characterization of the Saccharomyces cerevisiae Spt4, Spt5, and Spt6 proteins suggested that these proteins act as transcription factors that modify chromatin structure. In this work, we report new genetic and biochemical studies of Spt4, Spt5, and Spt6 that reveal a role for these factors in transcription elongation. We have isolated conditional mutations in SPT5 that can be suppressed in an allele-specific manner by mutations in the two largest subunits of RNA polymerase II (Pol II). Strikingly, one of these RNA Pol II mutants is defective for transcription elongation and the others cause phenotypes consistent with an elongation defect. In addition, we show that spt4, spt5, and spt6 mutants themselves have phenotypes suggesting defects in transcription elongation in vivo. Consistent with these findings, we show that Spt5 is physically associated with RNA Pol II in vivo, and have identified a region of sequence similarity between Spt5 and NusG, an Escherichia coli transcription elongation factor that binds directly to RNA polymerase. Finally, we show that Spt4 and Spt5 are tightly associated in a complex that does not contain Spt6. These results, taken together with the biochemical identification of a human Spt4-Spt5 complex as a transcription elongation factor (Wada et al. 1998), provide strong evidence that these factors are important for transcription elongation in vivo.