Yorba Linda, CA (PRWEB) June 15, 2016
A large portion of the RNA transcribed in eukaryotic cells is rapidly degraded in the nucleus. A poly-adenylation complex that is separate from the canonical poly(A) machinery is involved in initiating 3´-5´ degradation of nuclear RNAs. This non-canonical poly(A) machinery, called the Trf4/5-Air1/2-Mtr4 or TRAMP complex, catalyzes the addition of 3-4 adenosines on target RNA 3´-ends. This tags the transcript for 3´-5´ exonuclease digestion by the nuclear RNA exosome, which can either degrade or trim the RNA in a way that depends on the presence of RNA structures or RNA-binding proteins.
Inactivating the nuclear exosome stabilizes these RNAs, which would otherwise be short-lived. The cellular polyadenylation that follows lengthens the oligo(A) tails to >30 adenosines. Most of these poly(A)+ 3´-ends occur from non-coding and pervasive RNA polymerase II (Pol II) transcripts undergoing transcription termination by the Nrd1-Nab3-Sen1 (NNS) complex. Mapping the precise 3´-ends of these unstable RNAs is made possible by 3´-sequencing of RNAs from exosome-inactivated cells—which yields a high-resolution view of NNS termination genome-wide. Researchers were surprised to discover that different NNS-dependent terminators exhibit substantial heterogeneity in the width of the termination window. Through further analysis of NNS-terminators with a narrow termination window, they found that a particular set of DNA-binding proteins cooperate with NNS by roadblocking Pol II to promote efficient transcription termination genome-wide.
The researchers used QuantSeq 3´ mRNA-Seq Library Prep Kits from Lexogen to multiplex more than 40 samples per sequencing lane and obtain between two-million and five-million reads per sample. Analyzing many different strains with various exosome and roadblocking factors inactivated, they demonstrated that inactivating roadblocks shifted the window of NNS termination downstream. Remarkably, they found that disabling NNS enabled elongation of Pol II through the same roadblocks—which helps explain how RNA processing signals control the outcome of collisions between Pol II and DNA binding proteins.
Lexogen is sponsoring a new, free educational webinar, “Mapping nuclear-exosome targeted poly(A) tails with 3´-RNA seq,” which will discuss practical considerations involved in preparing QuantSeq 3´-poly(A)+ libraries and in processing, mapping, and analyzing reads. Attendees will also learn how to cluster poly(A) tags and perform differential expression analysis on clusters, and perform different types of meta-site/pileup analyses. Continuing education credits will be offered through the PACE system.
The speaker is Kevin Roy, PhD, a postdoctoral scholar in the laboratory of Dr. Lars Steinmetz in the Department of Genetics at Stanford University. Dr. Roy received a PhD in molecular biology in the laboratory of Dr. Guillaume Chanfreau at the University of California, Los Angeles (UCLA). At UCLA, he studied environment-dependent RNA degradation pathways in yeast, and developed high-throughput methods to map poly(A)+ 3´-ends of short-lived RNA species. His interests include high-throughput systems genetics, with a focus on how genetic variation interacts with environment to produce phenotype. Dr. Roy’s complete bio is found on the LabRoots website.
The free webinar, hosted by LabRoots, will be presented on June 22, 2016, at 9 am Pacific Time/12 pm Eastern Time/6 pm Central European Time.
For full details about the event, continuing education credits, and free registration, click here.
Lexogen, Vienna, Austria, is a biotech company that offers proprietary expression profiling technologies that enable detailed analysis of the complete transcriptome and individual full-length RNAs of interest. The company was founded in 2007 with the support of private capital and public funds, and is supported by the Austrian Research Promotion Agency FFG, the Austria Wirtschaftsservice AWS, INiTS, and the Wirtschaftsagentur Wien. Half of its employees work in research and development. The company has a US subsidiary based in New Hampshire.
LabRoots is the leading scientific social networking website and producer of educational virtual events and webinars. Contributing to the advancement of science through content sharing capabilities, LabRoots is a powerful advocate in amplifying global networks and communities. Founded in 2008, LabRoots emphasizes digital innovation in scientific collaboration and learning, and is a primary source for current scientific news, webinars, virtual conferences, and more. LabRoots has grown into the world’s largest series of virtual events within the Life Sciences and Clinical Diagnostics community.