The limited differences between the genomes of very different species have led to the emerging recognition that biological diversity is likely to largely derive from the complexity of RNA. This is evident in the diverse ways that RNA molecules are generated and processed and the various ways in which mRNA expression is regulated.
Just as new methodologies drove a revolution in our understanding of the role of RNA in biology in the twentieth century, new high-throughput sequencing, bioinformatics and biochemical methods are now being applied to whole tissues and genetically defined systems to generate new insights into the role of RNA in biological systems.
Alternative splicing is one of the best-studied mechanisms by which RNA diversity is generated. Regulation of splicing allows a means of generating RNA variants that offer great biological variability.
Alternative polyadenylation is emerging as an important means for regulating 3′ UTRs, which in turn offer various means of regulating gene expression, including microRNA-mediated control of translation, RNA localization and turnover.
The regulation of RNA processing involves a host of regulatory RNA-binding proteins (RNABPs) that act according to their affinities for different RNA sequences and the local abundances of RNAs and proteins. Therefore, a combination of biochemistry and cell biology will be required to fully understand RNA regulation in mammalian cells.
Genome-wide analysis of RNA–protein interactions can be rigorously approached biochemically using HITS-CLIP, and methods like next-generation sequencing offer a powerful means of quantifying RNA differences to enumerate RNA diversity. Putting the two approaches together using bioinformatic tools allows genome-wide functional RNA maps to be generated, and hence new rules of RNA regulation to be discovered.
An increasing number of human diseases are being found to relate to targeting of RNABPs, either through their mutation, autoimmune targeting or sequestration by RNA expansions. Applying these same genome-wide analyses to tissues affected by human disease offers the possibility of gaining new insights into disease pathogenesis and targeted therapeutics.
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.
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We are grateful to members of the laboratory for thoughtful discussions. We apologize to the many colleagues whose interesting studies we reviewed but were unable to cite here due to space limitations. This work was supported by grants from the National Institute of Health and the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
A hybrid structure consisting of RNA and DNA in which RNA displaces a DNA strand to hybridize to its complementary DNA sequence. The formation of R-loops was a key method to define the relationship between genes and their RNA products.
- 'RNA World' hypothesis
A hypothesis that life originated as an RNA-based form, based on the finding that RNA can act as both genetic material and an enzyme.
- Piwi-interacting RNA
A small RNA species that is processed from a single-stranded precursor RNA. They are 25–35 nucleotides in length and form complexes with the piwi protein. piRNAs are thought to have roles in transposon silencing and stem cell function.
- RNA editing
The post-transcriptional modification of RNA primary sequence by the insertion and/or deletion of specific bases, or the chemical modification of adenosine to inosine or cytidine to uridine.
- Exon-junction microarray
A microarray platform that contains probe sets designed to detect the mRNA sequences (junctions) formed by the splicing of one exon to another.
- Ultraconserved element
A large sequence in the genome (usually >200 nucleotides) that shows high levels of conservation across multiple species.
- Nonsense-mediated decay
The process by which mRNAs containing premature termination codons are destroyed to preclude the production of truncated and potentially deleterious protein products. It is also used in combination with specific alternative splicing events to control the levels of some proteins.
An oligomer of 25 nucleotides with bases linked to a morpholine ring. The oligomers can bind and inactivate selected RNA sequences on the basis of base pairing and steric interference.
- Small interfering RNA
RNA molecules that are 21–23 nucleotides long and that are processed from long double-stranded RNAs. They are functional components of the RNAi-induced silencing complex. They typically target and silence mRNAs by binding perfectly complementary sequences in the mRNA and causing their degradation and/or translation inhibition.
- Exon skipping
Exclusion of an exon from the resulting mature mRNA due to direct splicing of the upstream exon to the downstream exon.
- Seed site
A short RNA sequence that is bound by and necessary for microRNA-mediated RNA regulation.
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Licatalosi, D., Darnell, R. RNA processing and its regulation: global insights into biological networks. Nat Rev Genet 11, 75–87 (2010). https://doi.org/10.1038/nrg2673
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