Rtp1p Is a Karyopherin-Like Protein Required for RNA Polymerase II Biogenesis

Abstract

The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phenylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex. In eukaryotes, RNA polymerase II (RNA pol II) synthesizes mRNA and several noncoding RNAs. RNA pol II is a multisubunit enzyme composed of 12 subunits that are highly conserved among eukaryotes and whose structure has been well characterized (1). Yeast RNA pol II can dissociate into a 10-subunit catalytic core and a heterodimer of subunits Rpb4p and Rpb7p, which are required for the initiation from promoter DNA (2). The two large subunits, Rpb1p and Rpb2p, form the central mass of the enzyme, whereas the smaller subunits generally bind on the surface. Although the structure and functional regulation of RNA pol II have been characterized in detail, the study of its biogenesis has only recently been pursued. By assuming that the assembly of eukaryotic RNA pol II is similar to that of the bacterial polymerase, as subunit dissociation experiments suggest (3), it has been proposed that RNA pol II assembly would start with the formation of the Rpb3 subassembly, followed by the docking of the Rpb2 subassembly and finally the binding of the Rpb1 subassembly (4). The assembly process requires the participation of proteins that are not components of the mature enzyme. Several proteins that interact transiently with RNA pol II with a putative role as assembly factors have been identified. Thus, multiple interactions between GPN1/RPAP4 (Npa3p in yeast) and RNA pol II subunits in the soluble fraction of human cell extracts have been identified by performing protein affinity purification coupled to mass spectrometry (AP-MS) (5). GPN1 is a cytoplasmic-nuclear shuttling GTPase (5, 6) that associates with RNA pol II in a GTP-dependent manner (7). The involvement of GPN1 and its Saccharomyces cerevisiae homolog, Npa3p, in the nuclear import of RNA pol II has been confirmed by other reports (7, 8). Using MS-based quantitative proteomics on human cell extracts, a cytoplasmic RNA pol II intermediate containing the Rpb1p and Rpb8p subunits associated with th...

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.…”

Rbs1, a New Protein Implicated in RNA Polymerase III Biogenesis in Yeast Saccharomyces cerevisiae

“…Npa3, the yeast homolog of GPN1, is required for nuclear localization of yeast Pol II and binds it in a GTP-dependent manner (13), which argues that the mechanism involved in the subcellular localization of Pol II requires the catalytic function of GNP proteins and is conserved from yeast to mammals (14). Two other proteins involved in Pol II biogenesis, Iwr1 and Rtp1, were identified in genetic screens for suppressors of the growth defect caused by depletion of NC2, a negative regulator of mRNA transcription (15,16). Iwr1 binds yeast Pol II in the active-center cleft between the two largest subunits, possibly facilitating or sensing complete Pol II assembly in the cytoplasm.…”

“…Rtp1 interacts, to different extents, with several Pol II subunits and with members of the R2TP complex. Besides its role in subunit assembly, Rtp1, which is a karyopherin-like protein, is likely involved in nuclear transport of Pol II (16).…”

SKA1 induces de novo MTX‐resistance in osteosarcoma through inhibiting FPGS transcription

“…RPB3 is part of the core element within the central large cleft and the clamp element that moves to open and close this central large cleft. RPB11/POLR2J and RPB3/POLR2C subunits interact with each other . The MTX‐mediated interaction of RPB3 with SKA1 raises a possibility that SKA1 may interfere with RPB3 function and thus inhibits transcription of target genes, including FPGS.…”

“…RPB3-dependent transactivation of FPGS is suppressed by SKA1 in SF-86 cells DNA-dependent RNA polymerase II (Pol II) complex consists of many subunits. RPB3 is part of the core element within the central large cleft and the clamp [31]. The MTX-mediated interaction of RPB3 with SKA1 raises a possibility that SKA1 may interfere with RPB3 function and thus inhibits transcription of target genes, including FPGS.…”