Structure of a specific nuclear mRNP and the role of Hpr1 in mRNP assembly

Abstract

In eukaryotes, pre-mRNAs are processed and packaged by numerous RNA-binding proteins (RBPs) into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Mature mRNPs are exported to the cytoplasm for the translation into proteins. The TREX complex plays an important role in coupling transcription and mRNA export. In S. cerevisiae, it consists of the pentameric THO complex (Hpr1, Tho2, Mft1, Thp2 and Tex1), the helicase Sub2, the mRNA export adaptor Yra1, and the SR-like proteins Gbp2 and Hrb1. Deletion of THO component HPR1 results in vast consequences for the cell, such as increased genomic instability, hyperrecombination, accumulation of R-loops and an mRNA export defect. The THO complex interacts with Sub2, which loads Yra1 onto the mRNP for further recruitment of general mRNA exporter Mex67-Mtr2. Mex67 can be recruited to the mRNP through different adaptor proteins - Yra1, Nab2, Npl3 and Hpr1. The mRNA exporter Mex67-Mtr2 mediates the export of the mRNP through the nuclear-pore complex (NPC). The first aim of this study was to determine the structure of nuclear transcript-specific mRNPs. Isolation of a specific mRNPs was accomplished by a two-step affinity purification. In the first step, nuclear mRNPs were enriched via Cbc2, a subunit of the cap-binding complex. In the second step, RNA-based approaches using different types of antisense oligonucleotides and RNA aptamers were implemented to specifically purify CCW12 mRNA with its associated proteins. MS2 and Mango aptamers provided specific purification of the CCW12 mRNP in sufficient yield. Particles isolated via these aptamers were visualised by electron microscopy. This study demonstrates that specific mRNPs can be enriched in a good yield for further structural analysis using advanced electron microscopic techniques. Another aim of this study was to investigate the role of Hpr1 in mRNP assembly. It shows that deletion of HPR1 leads to an increased level of Nab2, Yra1 and Mex67 in nuclear mRNPs. It is consistent with these proteins binding more mRNAs in ∆hpr1 strain. The occupancy of Nab2 and Yra1 at the transcribed genes is increased in ∆hpr1 cells, but not for Mex67. To investigate mRNA export in the absence of Hpr1, other mRNA export adaptors, such as Nab2, Yra1 and Npl3, were overexpressed in ∆hpr1 cells. The mRNA export defect of ∆hpr1 cells is suppressed by overexpression of Nab2 or Yra1. In both cases, restored mRNA export is associated with similar changes in mRNP composition: further increase of Nab2 and reduction of Mex67 to the WT level. In case of Yra1 overexpression in ∆hpr1 cells, Yra1 recruits or retains more Nab2 at transcribed genes. Taken together, in cells lacking a functional THO complex, Yra1 can act as assistant to facilitate the loading of Nab2 onto mRNPs; high levels of Nab2 can promote mRNA export; increase of Mex67 amount is associated with nuclear mRNA retention, while reduction of Mex67 to the WT level is related to restored mRNA export. Deletion of another important component of mRNA export pathway, nucleoporin NUP60, leads to changes in nuclear mRNPs similar to a ∆hpr1 strain: increased levels of Nab2, Yra1 and Mex67. Therefore, it is hypothesized that increased levels of these proteins are hallmarks of mRNP retention caused by disruption of the mRNA export pathway.