JUN 22, 2016 09:00 AM PDT

Mapping nuclear-exosome targeted poly(A) tails with 3´-RNA seq

SPONSORED BY: Lexogen, Lexogen
C.E. CREDITS: P.A.C.E. CE
Speakers
  • Postdoctoral Scholar, Department of Genetics, Stanford University
    Biography
      Kevin obtained his Ph.D. at UCLA in the laboratory of Guillaume Chanfreau, where he studied RNA biology and developed high-throughput sequencing methods to map RNA degradation intermediates genome-wide. He is currently a National Research Council (NRC) postdoctoral associate in the laboratories of Dr. Lars Steinmetz at Stanford University and Dr. Marc Salit at the the Joint Initiative for Metrology in Biology (JIMB), a joint institute between Stanford University and the National Institute of Standards and Technology (NIST). Kevin works on developing high-throughput precision genome editing technologies with CRISPR/Cas9 to enable dissecting the genetic architecture underlying complex cellular phenotypes.

    Abstract:
    DATE: June 22, 2016
    TIME: 9am Pacific time, 12pm Eastern time, 6pm Central European time


    A large fraction of the RNA transcribed in eukaryotic cells is rapidly degraded in the nucleus. A poly-adenylation complex distinct from the canonical poly(A) machinery is responsible for initiating 3´-5´ degradation of nuclear RNAs. This non-canonical poly(A) machinery, termed 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 manner dependent on the presence of RNA structures or RNA-binding proteins. Inactivating the nuclear exosome stabilizes these otherwise short-lived RNAs, and subsequent cellular polyadenylation lengthens the oligo(A) tails to >30 adenosines.The majority of these poly(A)+ 3´-ends arise from non-coding and pervasive RNA polymerase II (Pol II) transcripts undergoing transcription termination by the Nrd1-Nab3-Sen1 (NNS) complex. 3´-sequencing of RNAs from exosome-inactivated cells enabled mapping the precise 3´-ends of these unstable RNAs, providing a high-resolution view of NNS termination genome-wide.Surprisingly, different NNS-dependent terminators display substantial heterogeneity in the width of the termination window, with some genes terminating the majority of transcripts in a window of <10 bp while others exhibit termination sites over a broad region of >500 bp. Further analysis of NNS-terminators with a narrow termination window revealed that a particular set of DNA-binding proteins cooperate with NNS by roadblocking Pol II to promote efficient transcription termination genome-wide. Using the QuantSeq 3´ mRNA-Seq library prep kits, we were able to multiplex >40 samples per sequencing lane and obtain between 2 to 5 million reads per sample. This enabled us to analyze numerous different strains with various exosome and roadblocking factors inactivated, showing that inactivating roadblocks shifted the window of NNS termination downstream. Strikingly, disabling NNS enabled elongation of Pol II through the same roadblocks.These results explain how RNA processing signals control the outcome of collisions between Pol II and DNA binding proteins.
     
    Learning Objectives:
    • learn practical considerations involved in preparing QuantSeq 3´-poly(A)+ libraries and in processing, mapping, and analyzing reads
    • learn how to cluster poly(A) tags and perform differential expression analysis on clusters, and perform different types of meta-site/pileup analyses

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