Regulatory mechanisms of incomplete huntingtin mRNA splicing

This review first discusses ways in which we can evaluate transcription inhibition, describe changes in nuclear structure due to transcription inhibition, and report on genes that are paradoxically stimulated by transcription inhibition. Next, it summarizes the characteristics and mechanisms of commonly used inhibitors: α-amanitin is highly selective for RNAP II and RNAP III but its action is slow, actinomycin D is fast but its selectivity is poor, CDK9 inhibitors such as DRB and flavopiridol are fast and reversible but many genes escape transcription inhibition. New compounds, such as triptolide, are fast and selective and able to completely arrest transcription by triggering rapid degradation of RNAP II.

Summary Transcription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5′splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.

DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.