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mRNA Editing, Processing and Quality Control in Caenorhabditis elegans - PubMed

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mRNA Editing, Processing and Quality Control in Caenorhabditis elegans

Joshua A Arribere et al. Genetics. 2020 Jul.

Abstract

While DNA serves as the blueprint of life, the distinct functions of each cell are determined by the dynamic expression of genes from the static genome. The amount and specific sequences of RNAs expressed in a given cell involves a number of regulated processes including RNA synthesis (transcription), processing, splicing, modification, polyadenylation, stability, translation, and degradation. As errors during mRNA production can create gene products that are deleterious to the organism, quality control mechanisms exist to survey and remove errors in mRNA expression and processing. Here, we will provide an overview of mRNA processing and quality control mechanisms that occur in Caenorhabditis elegans, with a focus on those that occur on protein-coding genes after transcription initiation. In addition, we will describe the genetic and technical approaches that have allowed studies in C. elegans to reveal important mechanistic insight into these processes.

Keywords: Caenorhabditis elegans; RNA editing; RNA modification; WormBook; polyadenylation; quality control; splicing.

Copyright © 2020 by the Genetics Society of America.

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Figures

Figure 1
Figure 1

Commonly used terms in the study of mRNA biogenesis and regulation. An mRNA begins its life with transcription and initially exists as a premature mRNA (pre-mRNA). The pre-mRNA includes more sequence than the mature mRNA will contain. The pre-mRNA is processed, spliced, and edited to give rise to the mature mRNA. Several commonly described features of the mature mRNA are indicated, including the trimethylguanosine “cap,” spliced leader (blue, which is appended after excision of the outron), 5′ UTR (the portion of the mRNA upstream of the start codon), CDS (green, coding DNA sequencing also called open reading frame (ORF), 3′ UTR (yellow, the region of mRNA downstream of the stop codon) and untemplated poly(A) tail. For simplicity, the pre-mRNA and mRNA are shown as discrete entities, though there is evidence that mRNA maturation occurs cotranscriptionally. The mature mRNA is exported from the nucleus, possibly translated, and eventually degraded.

Figure 2
Figure 2

The impact of ADARs on RNA. ADARs use water (H2O) to catalyze the removal of an amine group (red) from adenosine, resulting in inosine. While adenosine in RNA base-pairs with uracil, inosine base-pairs with cytosine.

Figure 3
Figure 3

Substrate recognition by the C. elegans ADARs. C. elegans

ADR-1

(blue) contains two dsRNA binding domains (ovals) and a deaminase domain (red), but lacks critical amino acids to perform deamination.

ADR-1

interacts physically with both

ADR-2

(red) and target mRNAs (teal/purple dsRNA) to promote editing by

ADR-2

at specific sites.

Figure 4
Figure 4

Schematic representations of trans-splicing and cis-splicing in C. elegans. (A) Spliced leader trans-splicing. A 2,2,7-trimethylguanosine (TMG)-capped 22-nt spliced leader (SL) sequence derived from an SL snRNA in SL snRNP replaces a 7-methylguanosine (m7G)-capped outron in a pre-mRNA. A y-shaped outron is excised. (B) Cis-splicing. A lariat-shaped intron is excised and the upstream and downstream exons are ligated. The 5′-splice site (5′SS), 3′-splice site (3′SS) and branch point (BP) are recognized by U1 snRNP (U1), U2 auxiliary factor (U2AF) and U2 snRNP (U2), respectively. Boxes represent exons and solid lines indicate introns and outrons. Dashed lines connect exons that are ligated in the splicing reactions. Cap structures and branch points are indicated with green and black circles, respectively. Almost invariable nucleotide sequences of the splice sites are indicated.

Figure 5
Figure 5

Sequence motifs of the 5′ (A) and 3′ (B) splice sites. Probability of the nucleotides at each position is displayed by using Weblogo version 3.7.1 (Crooks et al. 2004). The sequences of the introns are derived from 114,417 (5′) and 114,006 (3′) unique splice sites in 20-nt or longer introns annotated in WormBase (WS254). Position 0 indicates the beginning (A) and the end (B) of the introns.

Figure 6
Figure 6

Schematic representations of elementary alternative splicing events. (A) Cassette exon. (B and C) Competitive 5′- (B) and 3′- (C) splice sites. (D) Mutually exclusive exons. (E) Retained intron. (F) Back splicing. (G) Alternative first exons. The first exon of an mRNA is selected by alternative promoters. TSS, transcription start site. (H) Alternative last exons. The last exon of an mRNA is selected in conjunction with alternative polyadenylation sites. CPS, cleavage and polyadenylation site.

Figure 7
Figure 7

Schematic representations of core sequence elements and factors involved in cleavage and polyadenylation of mRNAs in mammals. Red arrowhead indicates CPS. CA, CA dinucleotide immediately 5′ to the CPS; CTD, C-terminal domain of Pol II; CF Im, mammalian cleavage factor I; CF IIm, mammalian cleavage factor II; CPSF, cleavage and polyadenylation specificity factor; CstF, cleavage stimulation factor; PAP, poly(A) polymerase; U, U-rich upstream element (USE); UGUA, upstream elements with UGUA consensus.

Figure 8
Figure 8

Schematic representations of cis-elements for 3′ end processing of mRNAs in C. elegans. (A) Typical cleavage and polyadenylation signals. AAUAAA or related sequences embedded in a U-rich region functions as a polyadenylation signal (PAS). CPS, cleavage and polyadenylation site; DSE, downstream element; TES, transcription end site; USE, upstream element. (B) Overlapping end region (ORE). Two closely located (11–39 nt apart) CPSs have their own PAS and share a U-rich region as either DSE or USE. (C) Tandem cleavage and polyadenylation sites (CPSs). Two or more PASs and CPSs are located in the same last exon but are ≥ 40 nt apart. (D) Alternative last exons (ALEs). Choice of the CPS is coupled with choice of the last exon of an mRNA. Boxes represent exons and solid lines indicate introns or 3′ flanking regions.

Figure 9
Figure 9

Translational surveillance pathways under consideration. (A) Translation of a normal mRNA. Ribosomes (gray) load near the trimethylguanosine cap (m2,2,7G) and locate a start codon (green stoplight). Ribosomes elongate until they terminate at a stop codon (red octagon). Upon termination, protein is released, and ribosomes are recycled for further rounds of translation. (B) Nonsense-mediated mRNA decay (NMD) arises from translation termination at a premature stop codon. The mRNA is destabilized through the action of at least seven SMG proteins. (C) Nonstop Decay arises from translation to the 3′ end of an mRNA, which can arise from mRNA cleavage (left) or polyadenylation upstream of a stop codon (right). The mRNA and nascent protein are degraded, and the ribosome is rescued. (D) No-Go Decay arises when a ribosome stalls as a result of a roadblock during elongation (yellow triangle). Such roadblocks include RNA hairpins, rare codons, and polybasic (Arg or Lys) amino acid runs. As with Nonstop Decay, the mRNA and nascent protein are degraded, and the ribosome is rescued.

Figure 10
Figure 10

Schematic of mRNAs that do, or do not, trigger NMD. For each gene, the wild-type allele is diagrammed above a mutant allele. In the diagrams, exons are indicated by boxes, coding regions by thicker boxes, stop codons by red octagons and exon-exon junctions by white gaps. The scale bar shows 100 or 1000 nt for each mRNA. The far-right column indicates whether the allele is an NMD target or not. Examples were chosen to illustrate different classes of NMD targets and are not exhaustive (see text for further examples). Annotations were taken from Ensembl, with 3′ UTR annonations from (Jan et al. 2011). (A) An example of a premature stop codon upstream of exon-exon junctions.

lin-29

(

n546

) is a smg-suppressible allele of

lin-29

(Hodgkin et al. 1989) that encodes an Arg > Stop mutation in the

lin-29

ORF (Rougvie and Ambros 1995). (B) An example of an NMD target from a mono-exonic gene.

dpy-5

(

e61

) is a Gly > Stop mutation (Thacker et al. 2006) that confers a smg-suppressible Dpy phenotype (Hodgkin et al. 1989). (C) An example of an allele that converts a normal stop codon to a premature stop codon.

unc-54

(

r293

) is a 256 bp deletion spanning the

unc-54

3′ UTR and poly(A) site that results in a fusion of the

unc-54

transcript with the downstream

aex-5

transcript. The

unc-54

(

r293

) allele is smg-suppressible (Hodgkin et al. 1989). The exact positions of the exon-exon junctions in the

unc-54

(

r293

) transcript are not known; the diagram represents a “best guess” based on the work of Pulak and Anderson (1993), Loepold and Ahmed (2014) and the

aex-5

transcript. (D) An example of an allele that creates premature stop codons but does not elicit NMD.

unc-73

(

e936

) is a splice site mutation that leads to usage of two cryptic splice sites (−1 and +23 nt relative to the normal splice site) generating out-of-frame premature stop codons. Neither transcript is an NMD target, though the reasons for this are not known (Roller et al. 2000).

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