Cooperative Interaction of Transcription Termination Factors with the RNA Polymerase II C-terminal Domain

Phosphorylation of the C-terminal domain of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTDinteracting domains that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CTD-interacting domains of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single amino acid mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′-end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Serine 2 phosphorylation density is highest

Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress

RNA-binding proteins play a key role in shaping gene expression profiles during stress, however, little is known about the dynamic nature of these interactions and how this influences the kinetics of gene expression. To address this, we developed kinetic cross-linking and analysis of cDNAs (\u3c7CRAC), an ultraviolet cross-linking method that enabled us to quantitatively measure the dynamics of protein\u2013RNA interactions in vivo on a minute time-scale. Here, using \u3c7CRAC we measure the global RNA-binding dynamics of the yeast transcription termination factor Nab3 in response to glucose starvation. These measurements reveal rapid changes in protein\u2013RNA interactions within 1\u2009min following stress imposition. Changes in Nab3 binding are largely independent of alterations in transcription rate during the early stages of stress response, indicating orthogonal transcriptional control mechanisms. We also uncover a function for Nab3 in dampening expression of stress-responsive genes. \u3c7CRAC has the potential to greatly enhance our understanding of in vivo dynamics of protein\u2013RNA interactions

Transcriptome-Wide Binding Sites for Components of the Saccharomyces cerevisiae Non-Poly(A) Termination Pathway: Nrd1, Nab3, and Sen1

RNA polymerase II synthesizes a diverse set of transcripts including both protein-coding and non-coding RNAs. One major difference between these two classes of transcripts is the mechanism of termination. Messenger RNA transcripts terminate downstream of the coding region in a process that is coupled to cleavage and polyadenylation reactions. Non-coding transcripts like Saccharomyces cerevisiae snoRNAs terminate in a process that requires the RNA–binding proteins Nrd1, Nab3, and Sen1. We report here the transcriptome-wide distribution of these termination factors. These data sets derived from in vivo protein–RNA cross-linking provide high-resolution definition of non-poly(A) terminators, identify novel genes regulated by attenuation of nascent transcripts close to the promoter, and demonstrate the widespread occurrence of Nrd1-bound 3′ antisense transcripts on genes that are poorly expressed. In addition, we show that Sen1 does not cross-link efficiently to many expected non-coding RNAs but does cross-link to the 3′ end of most pre–mRNA transcripts, suggesting an extensive role in mRNA 3′ end formation and/or termination

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Loss of the yeast SR protein Npl3 alters gene expression due to transcription readthrough

Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression

Genome-Wide Distribution of RNA-DNA Hybrids Identifies RNase H Targets in tRNA Genes, Retrotransposons and Mitochondria

During transcription, the nascent RNA can invade the DNA template, forming extended RNA-DNA duplexes (R-loops). Here we employ ChIP-seq in strains expressing or lacking RNase H to map targets of RNase H activity throughout the budding yeast genome. In wild-type strains, R-loops were readily detected over the 35S rDNA region, transcribed by Pol I, and over the 5S rDNA, transcribed by Pol III. In strains lacking RNase H activity, R-loops were elevated over other Pol III genes, notably tRNAs, SCR1 and U6 snRNA, and were also associated with the cDNAs of endogenous TY1 retrotransposons, which showed increased rates of mobility to the 5'-flanking regions of tRNA genes. Unexpectedly, R-loops were also associated with mitochondrial genes in the absence of RNase H1, but not of RNase H2. Finally, R-loops were detected on actively transcribed protein-coding genes in the wild-type, particularly over the second exon of spliced ribosomal protein genes