Actin and hnRNP U cooperate for productive transcription by RNA polymerase II

Abstract

To determine the role of actin-ribonucleoprotein complexes in transcription, we set out to identify novel actin-binding proteins associated with RNA polymerase II (Pol II). Using affinity chromatography on fractionated HeLa cells, we found that hnRNP U binds actin through a short amino acid sequence in its C-terminal domain. Post-transcriptional gene silencing of hnRNP U and nuclear microinjections of a short peptide encompassing the hnRNP U actin-binding sequence inhibited BrUTP incorporation in vivo. In living cells, we found that both actin and hnRNP U are associated with the phosphorylated C-terminal domain of Pol II, and antibodies to actin and hnRNP U blocked Pol II-mediated transcription. Taken together, our results indicate that a general actin-based mechanism is implicated in the transcription of most Pol II genes. Actin in complex with hnRNP U may carry out its regulatory role during the initial phases of transcription activation.

B23 acts as a nucleolar stress sensor and promotes cell survival through its dynamic interaction with hnRNPU and hnRNPA1

“…hnRNPU-B23 interaction is mediated by histone-binding domain of B23 and RNA-binding domain of hnRNPU Both B23 and hnRNPU were known to contain functional domains, which account for their distinctive biochemical functions (Hingorani et al, 2000;Kukalev Figure 1 B23 interacts with hnRNPA1 and hnRNPU in the cytosol on ActD treatment. (a) HeLa cells stably expressing GFP were treated with the 0.1% DMSO vehicle, CHX (25 mg/ml) and ActD (1 mg/ml), respectively, followed by immunostaining with anti-B23 antibody (red).…”

“…Protein hnRNPU, another hnRNPs family member, is able to stabilize specific mRNAs by binding to their 3 0 -untranslated regions (UTRs) (Yugami et al, 2007). Moreover, hnRNPU has a regulatory function during the initial phases of transcription activation by affecting the activity of RNA Pol II (Kukalev et al, 2005).…”

Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

“…Nuclear ␤-actin associates with components of the ATP-dependent chromatin-remodeling complexes (Olave et al, 2002;Bettinger et al, 2004), with RNP particles (Percipalle and Visa, 2006), and with the three RNA polymerases in the eukaryotic cell nucleus, both in vitro and in vivo (Hu et al, 2004;Philimonenko et al, 2004;Kukalev et al, 2005;Wu et al, 2006). The above studies have shown that nuclear actin is a component of large protein complexes that include RNA polymerases, transcription elongation factors, and accessory splicing factors that can be recruited to the RNAPII carboxy terminal domain and hence to promoters.…”

hnRNP-U directly interacts with WT1 and modulates WT1 transcriptional activation

“…The endogenous proteins physically interact via the zinc-fingers of WT1 and the middle domain of hnRNP-U, the interaction is direct and hnRNP-U can inhibit WT1-mediated transcriptional activation. hnRNP-U has been shown to regulate splicing and RNA transport as well as transcription (Kanai et al, 2004;Kukalev et al, 2005). Similar roles have been proposed for WT1 (Davies et al, 1998;Ladomery et al, 1999;Niksic et al, 2004), so the interaction with hnRNP-U could potentially regulate any of these processes.…”

“…This transcriptional inhibition by hnRNP-U was shown to involve the middle domain of hnRNP-U, the same domain that associates with WT1, suggesting that the hnRNP-U:WT1 interaction may mediate transcriptional inhibition by bringing hnRNP-U into the proximity of RNA polymerase II via WT1-binding sites in the promoters of target genes. In addition, a search for novel actin-binding proteins associated with RNA polymerase II identified hnRNP-U (Kukalev et al, 2005) and showed that both actin and hnRNP-U associate with the phosphorylated form of RNA polymerase II and are implicated in the initial phases of transcription. In the present study, we also detected a protein that matched either g-or b-actin in the MS-FIT database as immunoprecipitating with WT1 (Table 2.1).…”