RNA processing and its regulation: global insights into biological networks

Abstract

In recent years views of eukaryotic gene expression have been transformed by the finding that enormous diversity can be generated at the RNA level. Advances in technologies for characterizing RNA populations are revealing increasingly complete descriptions of RNA regulation and complexity; for example, through alternative splicing, alternative polyadenylation and RNA editing. New biochemical strategies to map protein-RNA interactions in vivo are yielding transcriptome-wide insights into mechanisms of RNA processing. These advances, combined with bioinformatics and genetic validation, are leading to the generation of functional RNA maps that reveal the rules underlying RNA regulation and networks of biologically coherent transcripts. Together these are providing new insights into molecular cell biology and disease.

Figure 1. Alternative pre-mRNA processing permits a single gene to encode multiple mRNA isoforms

In this example a single gene generates pre-mRNAs that are alternatively processed to yield mRNA isoforms with different coding and 3′ untranslated regions. Alternative protein-coding regions are established through mutually exclusive splicing of ‘B’ and ‘C’ exons and selection of one of two possible 3′ terminal exons (‘D’ and ‘E’). Further mRNA diversification can result from alternative selection of poly(A) sites (pA) in the same 3′ terminal exon (pA2 vs pA3 in the ‘E’ exon) generating mRNA isoforms with a short or long 3′ UTR. Additional events (not shown) can further diversify the resulting mRNA pool including transcription initiation at an alternative promoter, selection of alternative 3′ or 5′ splice sites (which change exon length), intron retention, and RNA editing.

Figure 2. Coupling of RNA processing to alternative RNA regulation

A. Alternative polyadenylation can generate mRNAs with common and isoform-specific 3′ UTR sequences. Changes in 3′ UTR length can alter the repertoire of regulatory elements present in the UTR, such as miRNA target sequences, thus affecting the ability of the transcript to be subject to different forms of post-transcriptional regulation, in this case mRNA degradation. B. Alternative splicing can lead to coding frameshifts, resulting in the introduction of premature translation termination codon (PTC). The presence of a PTC triggers degradation of the mRNA by the NMD pathway, thus regulation of alternative pre-mRNA splicing can be used to control transcript abundance, as evidenced in the physiologic regulation of the RNABPs Ptbp1 and Ptbp2–,.

Figure 3. Synergies between methodologies lead to new rules of RNA regulation

Biochemical methods, exemplified by HITS-CLIP, can yield genome-wide footprints of direct RNA-protein interactions, but lack functional information. In contrast, microarrays or RNA-Seq are able to correlate differences in RNA profiles between tissues or genetic systems such as KO vs. WT animals, but cannot distinguish direct from indirect targets. Bioinformatic analysis can also be used to identify sequence features associated with specific RNA regulatory events, but still require biochemical validation of putative regulatory interactions. Overlaying these approaches can yield a powerful maps of functional RNA-protein interaction sites for Nova,, and Fox1/2,,. Two important maps can be derived from combining these approaches, one biologic (bottom right panel), one mechanistic (bottom left panels). An assessment of the directly regulated mRNAs can address the extent to which there is a biologically coherence to the set of target RNAs—for example, the first such assessment of a genome-wide, directly regulated validated set of targets revealed that Nova regulates RNAs encoding synaptic functions,,. Second, new rules of regulation can be derived from combining experiments to yield functional maps—for example it became apparent that the position of binding in a transcript determines the outcome of Nova or Fox1/2,, to enhance or inhibit alternative exon inclusion.

Figure 4. Synergies between methods lead to new rules of RNA regulation

Biochemical methods as exemplified by HITS-CLIP can yield genome-wide footprints of direct RNA–protein interactions but lack functional information. By contrast, microarrays or RNA–seq can correlate differences in RNA profiles between tissues or genetic systems, such as knockout (KO) and wild-type (WT) animals, but cannot distinguish direct from indirect targets. Bioinformatic analysis can also be used to identify sequence features associated with specific RNA regulatory events but still requires biochemical validation of putative regulatory interactions. Overlaying these approaches can yield powerful maps of functional RNA–protein interaction sites for neuro-oncological ventral antigen (Nova),, and FOX1/2 (REFS 72,90,139) proteins. Two important maps can be derived from combining these approaches; one biological (bottom right panel) and one mechanistic (bottom left panels). An assessment of the directly regulated mRNAs can address the extent to which there is a biological coherence to the set of target RNAs; for example, the first such assessment of a genome-wide, directly regulated validated set of targets revealed that Nova regulates RNAs that encode synaptic functions,,. In addition, new rules of regulation can be derived from combining experiments to yield functional maps; for example, it became apparent that the position of protein binding in a transcript determines whether Nova or FOX1/2 (REFS 69,72,90) binding enhances or inhibits the inclusion of alternative exons. m⁷G, 5′ cap; pA, poly(A) site.