Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2

Abstract

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.

Rtr1 Is a Dual Specificity Phosphatase That Dephosphorylates Tyr1 and Ser5 on the RNA Polymerase II CTD

“…Based on our observations, and previous work we propose that Rtr1 is recruited to PolII during import and assembly of the polymerase 34 in a form where enzymatic activity is limited. Since Rtr1 does not dephosphoryate peptides carrying only isolated Tyr1 and Ser5 phospho-marks, Rtr1 remains inactive during transcriptional initiation, capping and promoter clearance (Figure 5 top).…”

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

“…Notably, all three subunits of the TTT complex (TELO2, TTI1 and TTI2) did copurify with R2TP/PFDL subunits. As was the case in our initial experiments1819, protein components of all three RNAPs are among the strongest interactors of the R2TP/PFDL complex, as were a number of associated factors including RPAP2, SLC7A6OS and TANGO6, which have been shown to be involved in RNAP biogenesis and nuclear import333435.…”

Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

“…Our model does not include any nuclear roles of Npa3, although they may exist, because nucleocytoplasmic shuttling of GPN1/Npa3 was reported previously (7,13,21,52). However, GPN-loop GTPases lack a nuclear localization signal (NLS), and mutations of GPN2 or GPN3 cannot be rescued by the fusion of a NLS to Rpb3 (12), consistent with the main function of these enzymes being cytosolic.…”