Environmental influences on RNA processing: Biochemical, molecular and genetic regulators of cellular response

Abstract

RNA processing has emerged as a key mechanistic step in the regulation of the cellular response to environmental perturbation. Recent work has uncovered extensive remodeling of transcriptome composition upon environmental perturbation and linked the impacts of this molecular plasticity to health and disease outcomes. These isoform changes and their underlying mechanisms are varied-involving alternative sites of transcription initiation, alternative splicing, and alternative cleavage at the 3' end of the mRNA. The mechanisms and consequences of differential RNA processing have been characterized across a range of common environmental insults, including chemical stimuli, immune stimuli, heat stress, and cancer pathogenesis. In each case, there are perturbation-specific contributions of local (cis) regulatory elements or global (trans) factors and downstream consequences. Overall, it is clear that choices in isoform usage involve a balance between the usage of specific genetic elements (i.e., splice sites, polyadenylation sites) and the timing at which certain decisions are made (i.e., transcription elongation rate). Fine-tuned cellular responses to environmental perturbation are often dependent on the genetic makeup of the cell. Genetic analyses of interindividual variation in splicing have identified genetic effects on splicing that contribute to variation in complex traits. Finally, the increase in the number of tissue types and environmental conditions analyzed for RNA processing is paralleled by the need to develop appropriate analytical tools. The combination of large datasets, novel methods and conditions explored promises to provide a much greater understanding of the role of RNA processing response in human phenotypic variation. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

Keywords: RNA processing response; environmental perturbation; splicing QTL.

Figure 1:. Mechanistic details of RNA processing.

Isoform composition and complexity is regulated by core cellular machinery, DNA/RNA binding factors, and DNA/RNA regulatory sequences. A second layer of regulation is added by the dynamic mutational and modification events occurring at both RNA and protein levels. (A) Alternative transcription initiation is governed by transcriptional mechanisms regulating the usage of DNA promoters and transcription start sites. (B) Alternative splicing is governed by the spliceosome machinery and auxiliary splicing factors, which guide the recruitment of core snRNAs such as U1 and U2 to the nascent RNA molecule and direct 5′ or 3′ splice site selection. (C) Alternative cleavage and polyadenylation sites and factors direct decisions involved in 3′ end processing of nascent molecules.

Figure 2:. The diversity of RNA processing changes across environmental perturbations.

While each environmental context that has been studied so far shows substantial evidence for complex transcriptome changes across all RNA processing categories (grey boxes), each context also has characteristic global changes (dark colored boxes). These involve particular event types that are either (1) frequently observed to change, (2) change with a greater magnitude, or (3) change in a directional pattern after the environmental perturbation. Notably, retained introns are consistently enriched across all environmental perturbations, indicating that changes in environmental context affect splicing efficiency rather than only isoform choice. Global retention of annotated retained introns may serve as a sign that both regulated and canonically constitutive introns are being retained due to overall perturbation of the splicing process.

Figure 3:. The magnitude of RNA processing changes across different contexts.

(A) Any two isoforms for a given gene are often both expressed at high levels, such that an isoform excluding a cassette exon (grey) may be expressed at an equal proportion to an isoform including the exon (red). (B) Natural genetic variation impacting isoform composition often has a small but consistent effect upon relative isoform levels. Most QTLs detected with current methods are common variants acting in cis, which are presumed to be drifting under nearly neutral selection rather than strong selective pressures. (C) RNA processing responses to common environmental perturbations activating a cellular defense system (such as stimuli or stresses) often have moderate effect sizes. These changes are characterized by global shifts in the usage of given sites or exons, putatively regulated by differential expression or post-translational modification of trans regulatory factors. (D) RNA processing changes in proliferative cancer cells are often quite severe and widespread, coupled with and likely regulated by deleterious somatic mutations in core spliceosome machinery or splicing factors.

Figure 4:. Methods to assay isoform composition.

(A) Cap-analysis of gene expression (CAGE) protocols are commonly used to interrogate the location and usage of transcription initiation sites. The technique relies on capture of molecules with a 5′ 7-methylguanosine cap, commonly through selective biotinylation of the cap. Following selection, a 5′ adapter is added, containing a enzyme recognition site that allows cleavage of the cDNA at a given distance away from the adapter. The resulting product can be assayed through PCR and sequencing with either conventional or high-throughput methods. (B) Rapid analysis of cDNA ends (RACE) protocols allow the interrogation of the location and usage of mRNA cleavage and polyadenylation sites. Synthesis of cDNA is initiated by annealing of an oligo-dT primer that contains a known 3′ adapter sequence. Second strand synthesis is initiated using either a gene-specific or random primer and the resulting product can be assayed through PCR and sequencing with either conventional or high-throughput methods. (C) Splicing reporter assays are used to validate or interrogate sequences that affect splice site usage. A minigene construct containing either a real intron flanked by its corresponding exons or a synthetic construct encoding either luciferase, GFP or RFP is cloned into a reporter plasmid. The splicing of the introns in any of theses cases can be assayed using quantitative RT-PCR. For luciferase constructs, the increased splicing of intervening intron would result in increased luciferase production, which can be assayed using fluorescence based assays. Finally, the GFP/RFP construct often interrogates usage of individual splice sites, such that usage of an upstream splice site would encode GFP, while usage of the downstream splice site would encode RFP. The relative proportion of splice site usage can then be assayed by sorting cells based on markers for GFP and RFP. (D) Short reads from RNA-sequencing techniques can be used to identify and quantify RNA processing patterns across multiple event types. Quantification of whole isoforms or exons often rely on the quantification reads within all regions present in an isoform, while methods specifically interrogating alternative patterns rely on reads that are informative for usage of particular exons. The most informative reads are junction reads that are split between two exons and span the resulting junction. We note that reads for terminal exons could indicate either differential TSS or polyA site usage (for AFEs and ALEs, respectively) or differential splicing of terminal exons to internal exons.