B2 RNA and Alu RNA repress transcription by disrupting contacts between RNA polymerase II and promoter DNA within assembled complexes

Abstract

Noncoding RNAs (ncRNAs) are now recognized as transregulators of eukaryotic transcription, a role once attributed exclusively to protein factors. Two ncRNAs in mammalian cells have been shown to repress general mRNA transcription by RNA polymerase II (Pol II) in response to heat shock: mouse B2 RNA and human Alu RNA. B2 and Alu RNAs bind directly and tightly to Pol II and co-occupy the promoters of repressed genes along with the polymerase. Here, we identified the molecular mechanism by which mouse B2 RNA and human Alu RNA repress Pol II transcription. Biochemical assays to probe the network of protein-DNA interactions at the promoter revealed that B2 and Alu RNAs prevent Pol II from establishing contacts with the promoter both upstream and downstream of the TATA box during closed complex formation. Disruption of these contacts correlates with transcriptional repression. We conclude that B2 and Alu RNA prevent Pol II from properly engaging the DNA during closed complex formation, resulting in complexes with an altered conformation that are transcriptionally inert. In the absence of its normal contacts with the promoter, Pol II is likely held in these inactive complexes on DNA through interactions with promoter-bound TATA box-binding protein and transcription factor IIB.T ranscription is an intricate biological process in which DNA is copied into RNA; it is the critical first step in gene expression. In eukaryotes, the enzyme RNA polymerase II (Pol II) transcribes protein-encoding genes into mRNA with assistance of general transcription factors (GTFs; specifically TFIIA, TFIID, TFIIB, TFIIF, TFIIE, and TFIIH) that are thought to function at most promoters (1). Transcriptional regulation of specific genes occurs through the remarkably balanced interplay of auxiliary factors such as promoter-specific activators and repressors, coregulators, and chromatin-modifying complexes (1). Traditionally, it was thought that all of these factors were proteins, but now it is becoming clear that noncoding RNAs (ncRNAs) also play important roles in regulating transcription. Indeed, diverse ncRNAs have been identified as regulators of nearly every step in the process of mRNA transcription from controlling chromatin structure through regulating transcript elongation (2).Our laboratory reported that 2 ncRNAs, mouse B2 RNA and human Alu RNA, repress mRNA transcription by binding to Pol II during the cellular heat shock response (3, 4). B2 and Alu RNAs are transcribed by RNA polymerase III from short interspersed elements (SINEs) (5). Upon heat shock, the levels of B2 RNA and Alu RNA increase (6, 7) and they function as general repressors of mRNA transcription (3, 4). Biochemical experiments showed that B2 and Alu RNAs bind directly to core Pol II with low nM affinity (4, 8). Other SINE RNAs have been identified in mammalian cells, including mouse B1 RNA and human scAlu RNA (5), the latter of which is likely derived from cleavage of full-length Alu RNA (9). The biological functions for B1 and scAlu RNAs are not known. In vitro both of...

B2 RNA represses TFIIH phosphorylation of RNA polymerase II

“…11 Within these inhibited complexes, B2 RNA disrupts the contacts between Pol II and the promoter DNA. 13 Consistent with this in vitro model for repression, cellbased experiments have co-localized Pol II and B2 RNA at the promoter of the repressed actin gene, specifically after heat shock in mouse cells. 12 We wondered what effect heat shock and B2 RNA would have on phosphorylation of the CTD of Pol II.…”

“…Previously, we had found that B2 RNA incorporates into complexes at the promoters of genes and represses transcription by keeping the polymerase from engaging promoter DNA. 13 Moreover, in heat-shocked cells we observed co-localization of B2 RNA and Pol II at the actin promoter. 12 Here we found that upon heat shock, Ser5 phosphorylation sharply decreases at the actin promoter, while the levels of TFIIH and total Pol II decrease to a much lower extent.…”

LncRNAs have a say in protein translation

“…Interestingly, it has been shown that SINEB2 RNAs are transcribed by RNA Pol III upon heat shock and repress transcription by disrupting the binding of RNA polymerase II to certain gene promoters [11,12]. In the case of the spliced antisense Uchl1 lncRNA, which contains SINEB2 and SINEB1 repeats, an effect on Uchl1 mRNA levels was not observed.…”

Dynamics of the association of heat shock protein HSPA6 (Hsp70B’) and HSPA1A (Hsp70–1) with stress-sensitive cytoplasmic and nuclear structures in differentiated human neuronal cells

“…These perispeckles are sites of active mRNA transcription that have been characterized as Btranscription factories^ (Brown et al 2008;Spector and Lamond 2011;Rieder et al 2012;Rieder et al 2014). The unique localization of HSPA6, but not HSPA1A, to perispeckles, suggests that HSPA6 may be associated with the recovery of transcription in human neuronal cells after inhibition by stressful stimuli (Allen et al 2004;Hieda et al 2005;Espinoza et al 2007;Yakovchuk et al 2009). Our on-going experiments indicate that localization of HSPA6 to perispeckles is disrupted by application of a transcriptional inhibitor during the recovery phase.…”