Abstract
Nascent mRNAs produced by transcription in the nucleus are subsequently processed and packaged into mRNA ribonucleoprotein particles (messenger ribonucleoproteins (mRNPs)) before export to the cytoplasm. Here, we have used the poly(A)-binding protein Nab2 to isolate mRNPs from yeast under conditions that preserve mRNA integrity. Upon Nab2-tandem affinity purification, several mRNA export factors were co-enriched (Yra1, Mex67, THO-TREX) that were present in mRNPs of different size and mRNA length. High-throughput sequencing of the co-precipitated RNAs indicated that Nab2 is associated with the bulk of yeast transcripts with no specificity for different mRNA classes. Electron microscopy revealed that many of the mRNPs have a characteristic elongated structure. Our data suggest that mRNPs, although associated with different mRNAs, have a unifying core structure.
Highlights
The TREX complex belongs to these adaptors and is composed of transcription elongation (THO complex) and export factors (Sub2 and Yra1)
Nab2 interacts with Mlp1, a nuclear pore complex-associated protein at the nuclear basket involved in mRNP quality control [10], with Gfd1 and Gle1, factors involved in mRNA export [11], and with the Sac3-Thp1 complex, which plays a role in coupling transcription with mRNA metabolism [12]
The co-precipitation of ribosomal proteins by Nab2-Tandem Affinity Purification (TAP) could indicate that mRNAs associated with Nab2 may have pulled down polysomes
Summary
Growth Conditions and Cell Lysis—Yeast cells were grown at 30 °C in 2l YPD medium (2% yeast extract, 1% peptone, 2% glucose) to A600 3.5. Ground pellets were stored at Ϫ80 °C until purification was performed
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