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

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

Iwr1 directs RNA polymerase II nuclear import

RNA polymerase (Pol) II transcribes protein-coding genes in the nucleus of eukaryotic cells and consists of 12 polypeptide subunits. It is unknown how Pol II is imported into the nucleus. Here we show that Pol II nuclear import requires the protein Iwr1 and provide evidence for cyclic Iwr1 function. Iwr1 binds Pol II in the active center cleft between the two largest subunits, maybe facilitating or sensing complete Pol II assembly in the cytoplasm. Iwr1 then uses an N-terminal bipartite nuclear localization signal that is recognized by karyopherin alpha to direct Pol II nuclear import. In the nucleus, Iwr1 is displaced from Pol II by transcription initiation factors and nucleic acids, enabling its export and recycling. Iwr1 function is Pol II specific, transcription independent, and apparently conserved from yeast to human

Amino Acid Residues Required for Physical and Cooperative Transcriptional Interaction of STAT3 and AP-1 Proteins c-Jun and c-Fos▿

Cooperation between STAT3 and c-Jun in driving transcription during transfection of reporter constructs is well established, and both proteins are present on some interleukin-6 (IL-6) STAT3-dependent promoters on chromosomal loci. We report that small interfering RNA knockdown of c-Jun or c-Fos diminishes IL-6 induction of some but not all STAT3-dependent mRNAs. Specific contact sites in STAT3 responsible for interaction of a domain of STAT3 with c-Jun were known. Here we show that the B-zip domain of c-Jun interacts with STAT3 and that c-Jun mutation R261A or R261D near but not in the DNA binding domain blocks in vitro STAT3-c-Jun interaction and decreases costimulation of transcription in transfection assays. Cooperative binding to DNA of tyrosine-phosphorylated STAT3 and both wild-type and R261A mutant c-Jun was observed. Even c-Jun mutant R261D, which on its own did not bind DNA, bound DNA weakly in the presence of STAT3. We conclude that a functional interaction between STAT3 and c-Jun while bound to chromosomal DNA elements exists and is necessary for driving transcription on at least some STAT3 target genes. Identifying such required interactive protein interfaces should be a stimulus to search for compounds that could ultimately inhibit the activity of STAT3 in tumors dependent on persistently active STAT3

The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation

Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription

The Prefoldin Bud27 Mediates the Assembly of the Eukaryotic RNA Polymerases in an Rpb5-Dependent Manner

<div><p>The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of <em>BUD27</em> affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of <em>RPB5</em>. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between <em>BUD27</em>, <em>RPB5</em>, and <em>RPB6</em>. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.</p> </div