Nuclear networking fashions pre-messenger RNA and primary microRNA transcripts for function

Abstract

The expression of protein-coding genes is enhanced by the exquisite coupling of transcription by RNA polymerase II with pre-messenger RNA processing reactions, such as 5'-end capping, splicing and 3'-end formation. Integration between cotranscriptional processing events extends beyond the nucleus, as proteins that bind cotranscriptionally can affect the localization, translation and degradation of the mature messenger RNA. MicroRNAs are RNA polymerase II transcripts with crucial roles in the regulation of gene expression. Recent data demonstrate that processing of primary microRNA transcripts might be yet another cotranscriptional event that is woven into this elaborate nuclear network. This review discusses the extensive molecular interactions that couple the earliest steps in gene expression and therefore influence the final fate and function of the mature messenger RNA or microRNA produced.

Figure 1

Cotranscriptional RNA processing. (A) Cotranscriptional pre-mRNA processing. Exons are represented as thick solid lines and introns as thin lines. Dashed arrows depict interactions between proteins that might stabilize interactions or promote the corresponding reaction. (i) Capping. Near the transcription initiation site, RNAPII is phosphorylated (P) on Ser5, which results in recruitment and activation of the capping enzymes. The cap binding complex (CBC) subsequently binds to the 5′ cap. Splicing factors (SFs) and some components of the CPA machinery are recruited to gene promoters through interactions with RNAPII, as well as associated transcription factors. SFs also bind to exonic sequence enhancers, and interactions with the C-terminal domain (CTD) might stabilize complex formation. Export factors in conjunction with the THO complex form the transcription-export (TREX) complex, which is recruited to the nascent transcript through interactions with the CBC. (ii) Spliceosome assembly. Phosphorylation of RNAPII on Ser2 allows elongation into the gene body. The spliceosome assembles on the first intron; assembly is enhanced by binding of SR proteins and other splicing factors to both the nascent RNA and the CTD, serving to bring the first and second exons into close proximity. The exon-junction complex (EJC) is recruited by the splicing machinery and is deposited just upstream of the exon–exon junction. The TREX complex stably associates with nascent RNA owing to interactions with CBC, as well as SFs and/or the EJC. (iii) Splicing of the 3′-terminal exon and 3′-end formation. The final intron and the 3′-terminal exon have been transcribed, and splicing of the final intron is under way. The CPA signal has also been transcribed, and interactions with splicing factors bound to the terminal exon, as well as the CTD, enhance recruitment of additional components of the CPA machinery. The CPA machinery reciprocally stabilizes interaction of SFs with the 3′-terminal intron. The CPA machinery assembles on the CPA signal and will cleave the RNA to release the transcript from the DNA template after splicing of the last intron is complete. (iv) Export. The processed mRNA is exported to the cytoplasm. Note that many proteins deposited on the pre-mRNA during splicing remain associated with the mRNA in the cytoplasm and might affect downstream processes. (B) Cotranscriptional pri-miRNA processing. (i) Microprocessor recruitment. The Microprocessor, consisting of Drosha, DGCR8 and additional accessory proteins, can be recruited to the pri-miRNA by means of the promoter, through interaction with Ars2 (which binds to CBC), through interaction with auxiliary proteins that bind to the pri-miRNA terminal loop (see Table 1 for examples) or through other, currently unknown, mechanisms. Auxiliary proteins might also inhibit Microprocessor interaction with the pri-miRNA. Note that the indicated interactions might affect pri-miRNA processing for only some miRNAs, as indicated by dashed arrows. (ii) Microprocessor assembly and cleavage. Drosha cleaves at the base of the hairpin to release the pre-miRNA. For some pri-miRNAs, Ars2 might facilitate Drosha cleavage. Proteins that bind to the pri-miRNA terminal loop could also facilitate or inhibit pri-miRNA cleavage. Note that, for intronic miRNAs, Drosha association with the pri-miRNA might be enhanced by interaction with splicing factors, and the bridging of exons mediated by binding of SR proteins to the flanking exons and to the CTD (as demonstrated in Figure 1Aii) would allow exons to be efficiently spliced despite prior cotranscriptional cleavage of the intronic miRNA. (iii) Pre-miRNA release and exonuclease recruitment. Exonucleases are recruited to the newly generated ends of the flanking pri-miRNA sequences. 5′–3′ degradation of the 3′ segment of the pri-miRNA might lead to premature termination of intergenic pri-miRNA transcription by RNAPII. (iv) Export. The pre-miRNA is exported to the cytoplasm by the Exportin5–RanGTP complex. Note that proteins that bind the pri-miRNA terminal loop, such as KSRP, might remain associated with the pre-miRNA and affect downstream processes in the cytoplasm such as Dicer cleavage. Hypothetical CTD phosphorylation states are indicated by question marks as the effect of CTD phosphorylation on pri-miRNA processing has not yet been determined.