Rate of transcription elongation and sequence-specific pausing by RNA polymerase I directly influence rRNA processing.

One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA by RNA polymerase I (Pol I). Processing of the resultant rRNA begins cotranscriptionally, and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking because of limited in vivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-Seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I in vitro; however, transcription elongation by the mutant has not been characterized in vivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both in vitro and in vivo. NET-Seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences in vivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-Seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the ribosomal DNA, particularly at the 5' end of the 35S gene. This combination of in vitro and in vivo analyses of a Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient cotranscriptional rRNA processing and ribosome assembly.

Essential histone chaperones collaborate to regulate transcription and chromatin integrity.

Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.

Identification of a candidate genetic variant for the Himalayan color pattern in dogs.

Acromelanism is a temperature-dependent hypopigmentation pattern commonly manifested as the Himalayan coat color found in rabbits, rats, mice, minks, and gerbils, wherein the extreme "points" are dark and the torso is pale. It is known as the Siamese pattern in cats. Himalayan color is genetically determined by the allelic variant ch of the locus C, later identified as the tyrosinase gene TYR. The tyrosinase functions at the initial steps of melanin production, and alteration of its activity by sequence changes results in pigmentation defects in vertebrates. The presence of acromelanism in dogs has not been described until now. We analyzed a DNA sample of a dachshund with a unique coat color resembling the Himalayan type. Sequencing of the coding part of the TYR gene from the proband revealed a homozygous variant (c.230G > A) in exon 1, leading to an amino acid substitution (p.R77Q) in a conserved region of the protein. The proband's mother, which is black-and-tan, is a heterozygous carrier of the c.230A allele, while none of the 210 dogs of different breeds, unrelated to the proband, carried the c.230A allele. These results suggest that the identified sequence variant is likely the cause of the Himalayan coloration of the proband.

Spt6 Is Required for the Fidelity of Promoter Selection.

Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.

Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements.

BACKGROUND: There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-nine novel RNA binding proteins for dengue virus (DENV) were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. CONCLUSIONS/SIGNIFICANCE: The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps cellular proteins for efficient amplification.

Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I.

Spt6 (suppressor of Ty6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele, spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression of RRN3 rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA.

Functional divergence of eukaryotic RNA polymerases: unique properties of RNA polymerase I suit its cellular role.

Eukaryotic cells express at least three unique nuclear RNA polymerases. The selective advantage provided by this enhanced complexity is a topic of fundamental interest in cell biology. It has long been known that the gene targets and transcription initiation pathways for RNA polymerases (Pols) I, II and III are distinct; however, recent genetic, biochemical and structural data suggest that even the core enzymes have evolved unique properties. Among the three eukaryotic RNA polymerases, Pol I is considered the most divergent. Transcription of the ribosomal DNA by Pol I is unmatched in its high rate of initiation, complex organization within the nucleolus and functional connection to ribosome assembly. Furthermore, ribosome synthesis is intimately linked to cell growth and proliferation. Thus, there is intense selective pressure on Pol I. This review describes key features of Pol I transcription, discusses catalytic activities of the enzyme and focuses on recent advances in understanding its unique role among eukaryotic RNA polymerases.

Divergent contributions of conserved active site residues to transcription by eukaryotic RNA polymerases I and II.

Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G). Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps.

The SWI/SNF chromatin remodeling complex influences transcription by RNA polymerase I in Saccharomyces cerevisiae.

SWI/SNF is a chromatin remodeling complex that affects transcription initiation and elongation by RNA polymerase II. Here we report that SWI/SNF also plays a role in transcription by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. Deletion of the genes encoding the Snf6p or Snf5p subunits of SWI/SNF was lethal in combination with mutations that impair Pol I transcription initiation and elongation. SWI/SNF physically associated with ribosomal DNA (rDNA) within the coding region, with an apparent peak near the 5' end of the gene. In snf6Δ cells there was a ∼2.5-fold reduction in rRNA synthesis rate compared to WT, but there was no change in average polymerase occupancy per gene, the number of rDNA gene repeats, or the percentage of transcriptionally active rDNA genes. However, both ChIP and EM analyses showed a small but reproducible increase in Pol I density in a region near the 5' end of the gene. Based on these data, we conclude that SWI/SNF plays a positive role in Pol I transcription, potentially by modifying chromatin structure in the rDNA repeats. Our findings demonstrate that SWI/SNF influences the most robust transcription machinery in proliferating cells.

Yeast transcription elongation factor Spt5 associates with RNA polymerase I and RNA polymerase II directly.

Spt5 is a transcription factor conserved in all three domains of life. Spt5 homologues from bacteria and archaea bind the largest subunit of their respective RNA polymerases. Here we demonstrate that Spt5 directly associates with RNA polymerase (Pol) I and RNA Pol II in yeast through its central region containing conserved NusG N-terminal homology and KOW domains. Deletion analysis of SPT5 supports our biochemical data, demonstrating the importance of the KOW domains in Spt5 function. Far Western blot analysis implicates A190 of Pol I as well as Rpb1 of Pol II in binding Spt5. Three additional subunits of Pol I may also participate in this interaction. One of these subunits, A49, has known roles in transcription elongation by Pol I. Interestingly, spt5 truncation mutations suppress the cold-sensitive phenotype of rpa49Δ strain, which lacks the A49 subunit in the Pol I complex. Finally, we observed that Spt5 directly binds to an essential Pol I transcription initiation factor, Rrn3, and to the ribosomal RNA. Based on these data, we propose a model in which Spt5 is recruited to the rDNA early in transcription and propose that it plays an important role in ribosomal RNA synthesis through direct binding to the Pol I complex.

Non-Mendelian determinant [ISP+] in yeast is a nuclear-residing prion form of the global transcriptional regulator Sfp1.

Four protein-based genetic determinants or prions-[SWI(+)], [MCA], [OCT(+)], and [MOT3(+)]-are recent additions to the list of well-known Saccharomyces cerevisiae prions, [PSI(+)], [URE3], and [PIN(+)]. A rapid expansion of this list may indicate that many yeast proteins can convert into heritable prion forms and underscores a problem of prion input into cellular physiology. Here, we prove that the global transcriptional regulator Sfp1 can become a prion corresponding to the prion-like determinant [ISP(+)] described earlier. We show that SFP1 deletion causes an irreversible [ISP(+)] loss, whereas increased SFP1 expression induces [ISP(+)] appearance. Cells that display the [ISP(+)] phenotype contain the aggregated form of Sfp1. Indeed, these aggregates demonstrate a nuclear location. We also show that the phenotypic manifestation of Sfp1 prionization differs from the manifestation of SFP1 deletion. These properties and others distinguish [ISP(+)] from yeast prions described to date.