], were recently described (6, 7). Thus, it is evident that protein inheritance is a widespread phenomenon, at least in lower eukaryotes.The discovery of prions in yeast occurred in different ways. Some (i.e., [PSI + ] and [URE3]) were long known as genetic determinants of mysterious nature until their prion nature was proposed (8). The others were revealed by purposeful screening of potentially prionogenic proteins and corresponding determinants. The prion-like determinant [ISP + ], described in our earlier work (9), belongs to the first group, because it was detected as a nonchromosomal antisuppressor in strains containing specific sup35 nonsense suppressor mutations and the nonsense mutations his7-1 (UAA) and lys2-87 (UGA (Fig. 1A). The Sup + phenotype cosegregated with Ura + in tetrads of the diploid that was obtained by crossing the sfp1Δ and [ISP + ] strains (Fig. 1B). These findings indicate either that [ISP + ] is a prion form of Sfp1 or that the change in phenotype was caused by an independent manifestation of the SFP1-null allele.To distinguish between these two possibilities, the sfp1Δ strain was transformed with the centromeric vector pRS315-SFP1. Introduction of the wild-type SFP1 allele did not change the phenotype of the sfp1Δ strain [i.e., the absolute majority (556 of 559) of transformants has retained the Sup + phenotype]. This fact suggests that the change of phenotype in the sfp1Δ strain was caused by [ISP + ] loss rather than phenotypic effects of the SFP1 deletion; otherwise, restoration of the Sup − phenotype would be observed. Notably, this loss was irreversible, because we have not observed a single example of Sup -clones appearing in the mitotic progeny of sfp1Δ strains in contrast to [isp -] strains obtained by GuHCl treatment, which produced Sup -clones at a high frequency (9). These results confirmed that SFP1 could be considered as a likely host gene for [ISP + ].

SUMMARY 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.

SUMMARY 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 function, 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.

4 Co-first authors 5 Lead contact running head: Spt6 regulates transcription initiation keywords: Spt6; transcription start sites; intragenic promoters; chromatin structure correspondence: winston@genetics.med.harvard.edu 2 SUMMARY 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 and reveal the magnitude of the potential for expressing alternative genetic information via intragenic promoters.3

BackgroundThere 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 FindingsSeventy-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/SignificanceThe 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.