Numerous Post-translational Modifications of RNA Polymerase II Subunit Rpb4/7 Link Transcription to Post-transcriptional Mechanisms.

Rpb4/7 binds RNA polymerase II (RNA Pol II) transcripts co-transcriptionally and accompanies them throughout their lives. By virtue of its capacity to interact with key regulators (e.g., RNA Pol II, eIF3, and Pat1) temporally and spatially, Rpb4/7 regulates the major stages of the mRNA life cycle. Here we show that Rpb4/7 can undergo more than 100 combinations of post-translational modifications (PTMs). Remarkably, the Rpb4/7 PTM repertoire changes as the mRNA/Rpb4/7 complex progresses from one stage to the next. These temporal PTMs regulate Rpb4 interactions with key regulators of gene expression that control transcriptional and post-transcriptional stages. Moreover, one mutant type specifically affects mRNA synthesis, whereas the other affects mRNA synthesis and decay; both types disrupt the balance between mRNA synthesis and decay ("mRNA buffering") and the cell's capacity to respond to the environment. We propose that temporal Rpb4/7 PTMs mediate the cross-talk among the various stages of the mRNA life cycle.

Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.

Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹5N and ¹4N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.