Unknown,Transcriptomics,Genomics,Proteomics

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Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs

ABSTRACT: The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL_short RNA from the ferritin mRNA 5' UTR. Importantly, DIS3L2 contributes to surveillance of pre-snRNAs during their cytoplasmic maturation. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exosome-associated DIS3. Using 3' RACE-Seq, we demonstrate that all novel DIS3L2 substrates are uridylated in vivo by TUT4/TUT7 poly(U) polymerases. Uridylation-dependent DIS3L2-mediated decay can be recapitulated in vitro, thus reinforcing the tight cooperation between DIS3L2 and TUTases. Together these results indicate that catalytically inactive DIS3L2, characteristic of Perlman syndrome, can lead to deregulation of its target RNAs to disturb transcriptome homeostasis. To investigate DIS3L2 functions genome-wide, total RNA samples were collected from model cell lines producing either WT or mut DIS3L2 three days after induction with doxycycline. The RNA samples were rRNA-depleted before preparation of strand-specific total RNA libraries according to the standard TruSeq (Illumina) protocol. TruSeq library preparation favours RNA molecules longer than 200 nt, and shorter transcripts are suboptimal for sequencing via this protocol. Thus, to obtain information about potential DIS3L2 RNA substrates with lengths between 20 and 220 nt, another RNA-Seq was carried out in parallel (with size selection through gel purification). The stable inducible HEK293 cell lines producing DIS3L2 variants were obtained using âpAL_01â and âpAL_02â plasmid constructs and the Flp-Inâ¢ T-RExâ¢ system according to the manufacturerâs guidelines. âpAL_01â and âpAL_02â plasmids are vectors for co-expression of recoded C-terminal FLAG-tagged DIS3L2 [wild type (WT) variant or its catalytic mutant counterpart (mut), respectively] and sh-miRNAs directed against endogenous DIS3L2 mRNA.

ORGANISM(S): Homo sapiens

SUBMITTER: Tomasz Kulinski

PROVIDER: E-GEOD-82336 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.

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Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs

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