RNA-seq of Yra1 associated mRNAs

Gene expression results from mRNA - RNA-binding protein (RBP) interactions within messenger ribonucleoprotein (mRNP) particles. We identified heterogeniety of nuclear mRNP composition involved in mRNA biogenesis and export with different occupancy and stoichiometry of RNA-binding proteins. Especially, mRNA export adopter protein Yra1 showed large variation of its stoichiometry on single mRNPs. We identified stoichiometry of Yra1 in the nuclear cap binding complex (Cbp80) bound mRNPs and it can be dissected into two categories (one and multiple Yra1 bound mRNPs). Multiple Yra1 containing mRNPs specifically co-occupied with THO complex (Hpr1). To investigate gene dependency to form different Yra1 stoichiometry mRNPs, we performed RNA-IP experiment for Yra1 with two step purification by Cbp80/Yra1 and Hpr1/Yra1. Overall design: RNA-seq of Cbp80/Yra1 and Hpr1/Yra1 co-occupied mRNPs isolated by two-step pulldown of Cbp80/Yra1 and Hpr1/Yra1 as well as total RNA isolated from pre-pulldown cell lysate. For the first pulldown, mRNPs are isolated by protein A (PrA) tagged Cbp80 or Hpr1. No PrA tagged strain was used as negative control. The isolated mRNPs are further selected by Yra1 pulldown to analyze Yra1 bound mRNPs.