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The 5′ → 3′ exoribonuclease XRN1/Pacman and its functions in cellular processes and development

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Abstract XRN1 is a 5′ → 3′ processive exoribonuclease that degrades mRNAs after they have been decapped. It is highly conserved in all eukaryotes, including homologs in Drosophila melanogaster (Pacman), Caenorhabditis elegans (XRN1), and Saccharomyces cerevisiae (Xrn1p). As well as being a key enzyme in RNA turnover, XRN1 is involved in nonsense‐mediated mRNA decay and degradation of mRNAs after they have been targeted by small interfering RNAs or microRNAs. The crystal structure of XRN1 can explain its processivity and also the selectivity of the enzyme for 5′ monophosphorylated RNA. In eukaryotic cells, XRN1 is often found in particles known as processing bodies (P bodies) together with other proteins involved in the 5′ → 3′ degradation pathway, such as DCP2 and the helicase DHH1 (Me31B). Although XRN1 shows little specificity to particular 5′ monophosphorylated RNAs in vitro, mutations in XRN1 in vivo have specific phenotypes suggesting that it specifically degrades a subset of RNAs. In Drosophila, mutations in the gene encoding the XRN1 homolog pacman result in defects in wound healing, epithelial closure and stem cell renewal in testes. We propose a model where specific mRNAs are targeted to XRN1 via specific binding of miRNAs and/or RNA‐binding proteins to instability elements within the RNA. These guide the RNA to the 5′ core degradation apparatus for controlled degradation. WIREs RNA 2012, 3:455–468. doi: 10.1002/wrna.1109 This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Turnover and Surveillance > Regulation of RNA Stability RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development

Overview of the three pathways used in eukaryotes for mRNA degradation. The circular conformation of mRNA due to the 5′ cap interacting with the 3′ poly‐A tail can be disrupted by removal of the 5′ cap (decapping), removal of the 3′ poly‐A tail (deadenylation), or by endonucleolytic cleavage (e.g., due to RNAi, or nonsense‐mediated mRNA decay in some organisms). Decapping exposes the mRNA to degradation by the 5′ → 3′ exoribonuclease XRN1 (Pacman) and deadenylation allows access for 3′ → 5′ degradation by the exosome complex. Cleavage creates two fragments, each of which is susceptible to either XRN1 or the exosome.

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XRN1 is highly conserved between Drosophila melanogaster and Homo sapiens and is similar to XRN2 in the nuclease domain (green area), particularly within the two conserved regions (CR1 and CR2, orange areas). Four domains have been identified in the central region of D. melanogaster Pacman12 that are not present in XRN2, but can be found in H. sapiens XRN1. Amino acid sequences of the protein domains were compared using Vector NTI Advance 11 (Invitrogen, Carlsbad, California) with the percent similarity shown together with the percent identity (in parenthesis).

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Proposed model to explain degradation of specific transcripts by XRN1/Pacman. Transcripts targeted for degradation by the 5′ → 3′ pathway are bound by RNA‐binding protein (purple) and/or a miRNA (black) at an RNA instability element (red) within the 3′ UTR. This results in assembly of the 5′ → 3′ degradation complex. XRN1/Pacman (yellow) is recruited by the RNA‐binding protein and/or the miRNA and in turn recruits and activates the catalytic decapping protein Dcp2 (pink). The addition of other decapping activators including Me31B and Dcp1 completes the active complex and the target is degraded.

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pacman mutants display defects in testes morphology and sperm production. (a) The testes of young, hemizygous pcm5 and pcm3 males are significantly disrupted compared to wild type, due to a reduction in width (b). This results fewer sperm being produced (c).42

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Drosophila melanogaster pacman mutants display a number of developmental phenotypes.69 (a) A wild‐type fly thorax, which forms as the wing imaginal discs grow together and fuse along the dorsal midline during pupation. (b) A pcm5 fly displaying a ‘cleft thorax’ phenotype where the wing imaginal disc cells have failed to grow/migrate completely across the gap. (c) The wings of a wild‐type fly showing their natural iridescence. (d) A pcm5 fly with ‘dull’ wings that lack iridescence.

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(a) Hypomorphic pacman alleles were created by imprecise excision of the P‐element P{EP}1526 in D. melanogaster. Red lines represent deletions (516 bp in pcm5 and 1378 bp in pcm3). The red box for pcm5 represents a section of the pcm gene that is put out of frame by the deletion. (b) The level of Pcm protein is reduced in both whole male and female adults for both pcm3 and pcm5, with the pcm5 level being almost undetectable by Western blotting. However, genetic evidence shows the pcm5 allele is hypomorphic, so some partially functional protein must be produced.69 (c) The expression of mRNA from the pcm gene in pcm5 and pcm3 was compared to the wild‐type level in whole L3 larvae, using a TaqMan quantitative Reverse Transcription Polymerase Chain Reaction (Applied Biosystems, Foster City, California) designed at the interface between exons 3 and 4. The pcm mRNA in pcm3 (two biological replicates and six technical replicates) was the same as wild type, while the level in pcm5 was reduced by threefold (three biological replicates and nine technical replicates, P < 0.001). Error bars show standard error of the mean.

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xrn1 RNAi in Caenorhabditis elegans causes a lethal developmental phenotype. In wild‐type embryos (a) ventral enclosure completes after hypodermal cells migrate around and fuse on the ventral side. In xrn1 knockdown embryos (b), migration and sealing fail, which results in a ventral hole.67

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RNA Turnover and Surveillance > Regulation of RNA Stability
RNA in Disease and Development > RNA in Disease
RNA in Disease and Development > RNA in Development
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes

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