RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented.
Splicing regulates gene expression and contributes to proteomic diversity in higher eukaryotes. However, in yeast only 283 of the 6000 genes contain introns and their impact on cell function is not clear. To assess the contribution of introns to cell function, we initiated large-scale intron deletions in yeast with the ultimate goal of creating an intron-free model eukaryote. We show that about one-third of yeast introns are not essential for growth. Only three intron deletions caused severe growth defects, but normal growth was restored in all cases by expressing the intronless mRNA from a heterologous promoter. Twenty percent of the intron deletions caused minor phenotypes under different growth conditions. Strikingly, the combined deletion of all introns from the 15 cytoskeleton-related genes did not affect growth or strain fitness. Together, our results show that although the presence of introns may optimize gene expression and provide benefit under stress, a majority of introns could be removed with minor consequences on growth under laboratory conditions, supporting the view that many introns could be phased out of Saccharomyces cerevisiae without blocking cell growth.
The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space. Most notably, RPAP4/GPN1 silencing provokes the retention of RPAP2 in the nucleus. Our results support a model in which RPAP2 enters the nucleus in association with RNAP II and returns to the cytoplasm in association with the GTPase GPN1/RPAP4. Although binding of RNAP II to RPAP2 is mediated by an N-terminal domain (amino acids 1–170) that contains a nuclear retention domain, and binding of RPAP4/GPN1 to RPAP2 occurs through a C-terminal domain (amino acids 156–612) that has a dominant cytoplasmic localization domain. In conjunction with previously published data, our results have important implications, as they indicate that RPAP2 controls gene expression by two distinct mechanisms, one that targets RNAP II activity during transcription and the other that controls availability of RNAP II in the nucleus.
Over the past 3 decades, many efforts have been made to identify and characterize the factors that regulate the activity of RNA polymerase II (RNAPII), the enzyme that synthesizes all the mRNA and many small nuclear RNA in eukaryotes. Quite surprisingly, very little is known about the cell machinery that regulates the fate of RNAPII before or after transcription. In this study, we report that XAB1/GPN1, a member of a unique class of GTPases containing a conserved GPN‐loop, is involved in the biogenesis of RNAPII by regulating its nuclear import. XAB1/GPN1 is physically associated with RNAPII in the soluble cell fraction and is essential for cell growth. Accumulation of RNAPII in the cytoplasm of the cell was induced upon (i) depletion of XAB1/GPN1 by siRNA silencing, (ii) overexpression of a XAB1‐GFP dominant negative fusion protein, (iii) mutation of the GTP‐binding or GPN motifs of the XAB1/GPN1 homologue in yeast, NPA3, and (iv) treatment of cells with leptomycin B, an inhibitor of XPO1/CRM1‐dependent nuclear export. Yeast strains with mutations in the NPA3 gene showed hypersensitivity to benomyl, a specific inhibitor of microtubule assembly, suggesting a role for microtubules in RNAPII biogenesis. Together, these results indicate that XAB1/GPN1 shuttles between the cytoplasm and the nucleus to mediate nuclear import of RNAPII.
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