The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3′ ends

Xie, Mingyi; Zhang, Wei; Shu, Mei‐Di; Xu, Acer; Lenis, Diana A.; DiMaio, Daniel; Steitz, Joan A.

doi:10.1101/gad.266973.115

Cited by 47 publications

(42 citation statements)

References 56 publications

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“…3 snRNA with a poly-G tail at 3′ end ( pre-U2 . 3-pG) , which prevents 3′ trimming activity [33], to rule out the possibility that the product is generated from the 3′ end trimming rather than endonucleolytic cleavage. The accumulation of U2 .…”

Section: Resultsmentioning

confidence: 99%

snRNA 3′ End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis

Liu

Chen

et al. 2016

PLoS Biol

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Uridine-rich small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play essential roles in splicing. The biogenesis of the majority of snRNAs involves 3′ end endonucleolytic cleavage of the nascent transcript from the elongating DNA-dependent RNA ploymerase II. However, the protein factors responsible for this process remain elusive in plants. Here, we show that DEFECTIVE in snRNA PROCESSING 1 (DSP1) is an essential protein for snRNA 3′ end maturation in Arabidopsis. A hypomorphic dsp1-1 mutation causes pleiotropic developmental defects, impairs the 3′ end processing of snRNAs, increases the levels of snRNA primary transcripts (pre-snRNAs), and alters the occupancy of Pol II at snRNA loci. In addition, DSP1 binds snRNA loci and interacts with Pol-II in a DNA/RNA-dependent manner. We further show that DSP1 forms a conserved complex, which contains at least four additional proteins, to catalyze snRNA 3′ end maturation in Arabidopsis. The catalytic component of this complex is likely the cleavage and polyadenylation specificity factor 73 kDa-I (CSPF73-I), which is the nuclease cleaving the pre-mRNA 3′ end. However, the DSP1 complex does not affect pre-mRNA 3′ end cleavage, suggesting that plants may use different CPSF73-I-containing complexes to process snRNAs and pre-mRNAs. This study identifies a complex responsible for the snRNA 3′ end maturation in plants and uncovers a previously unknown function of CPSF73 in snRNA maturation.

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Section: Resultsmentioning

confidence: 99%

snRNA 3′ End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis

Liu

Chen

et al. 2016

PLoS Biol

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“…To corroborate the in vivo interaction, we visualized the METTL16-MALAT1 ENE+A RNP in HeLa cells by using an in situ RNA-proximity ligation assay (RNA-PLA) (15). RNA-PLA uses a cDNA oligonucleotide to recognize a target RNA and an antibody to recognize the protein factor of the putative RNP.…”

Section: Mettl16 Is An Abundant Nuclear Protein That Interacts With Mmentioning

confidence: 99%

“…Cellular debris was removed via centrifugation for 15 min at 15,000 × g and 4°C. One-third of the nuclear-enriched lysate was treated with 3 μg of protease-free RNase A (ThermoFisher) for 15 Immunofluorescence. HeLa cells were fixed onto chamber slides with 4% (vol/vol) paraformaldehyde in PBS for 30 min on ice, permeabilized with 0.2% Triton X-100 in PBS for 10 min on ice, and blocked with 5 mg/mL BSA in PBS for 1 h on ice.…”

Section: Emsa Rnas Were 5′-[mentioning

confidence: 99%

Methyltransferase-like protein 16 binds the 3′-terminal triple helix of MALAT1 long noncoding RNA

Brown

Kinzig

DeGregorio

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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211

205

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; reviewed by Brenda L. Bass and Eric M. Phizicky)Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3′-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U • A-U triples interrupted by a C • G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding. Native gel-shift assays revealed a shift in radiolabeled MALAT1 ENE+A RNA upon addition of HEK293T cell lysate. Competitive gel-shift assays suggested that protein binding depends not only on the triple-helical structure but also its nucleotide composition. Selection from the lysate using a biotinylated-RNA probe followed by mass spectrometry identified methyltransferase-like protein 16 (METTL16), a putative RNA methyltransferase, as an interacting protein of the MALAT1 ENE+A. Gel-shift assays confirmed the METTL16-MALAT1 ENE+A interaction in vitro: Binding was observed with recombinant METTL16, but diminished in lysate depleted of METTL16, and a supershift was detected after adding anti-METTL16 antibody. Importantly, RNA immunoprecipitation after in vivo UV cross-linking and an in situ proximity ligation assay for RNA-protein interactions confirmed an association between METTL16 and MALAT1 in cells. METTL16 is an abundant (∼5 × 10 5 molecules per cell) nuclear protein in HeLa cells. Its identification as a triple-stranded RNA binding protein supports the formation of RNA triple helices inside cells and suggests the existence of a class of triple-stranded RNA binding proteins, which may enable the discovery of additional cellular RNA triple helices.RNA binding protein | RNA triple helix | noncoding RNA

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“…This MFE fold, however, sequesters the HuR and Sm binding sites in structured regions (Lee et al 1988;Myer et al 1992); additionally, it does not contain the 3 ′ terminal hairpin shared by other HSURs that is a structural mimic of host snRNA 3 ′ hairpins (Murthy et al 1986;Lee et al 1988;Wassarman et al 1989;Albrecht and Fleckenstein 1992;Golembe et al 2005b). SnRNA 3 ′ terminal hairpins are necessary for the assembly of Sm proteins (Golembe et al 2005a,b) and may also be important for cleavage by the Integrator complex, which is responsible for the 3 ′ -end processing of snRNAs (Baillat et al 2005;Ezzeddine et al 2011;Xie et al 2015). Thus, this energetically stable, but biologically unrealistic model, seemed unlikely to represent the native in vivo fold of HSUR1.…”

Section: Hsur1 Adopts a Different Conformation Than Previously Predictedmentioning

confidence: 99%

Host miRNA degradation by Herpesvirus saimiri small nuclear RNA requires an unstructured interacting region

Pawlica

Moss

Steitz

2016

RNA

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Herpesvirus saimiri, an oncogenic herpesvirus, during latency produces seven small nuclear RNAs, called the Herpesvirus saimiri U RNAs (HSUR1-7). HSUR1 mediates degradation of the host microRNA, miR-27, via a process that requires imperfect basepairing. The decreased levels of miR-27 lead to prolonged T-cell activation and likely contribute to oncogenesis. To gain insight into HSUR1-mediated degradation of miR-27, we probed the in vivo secondary structure of HSUR1 and coupled this with bioinformatic structural analyses. The results suggest that HSUR1 adopts a conformation different than previously believed and that the region complementary to miR-27 lacks stable structure. To determine whether HSUR1 structural flexibility is important for its ability to mediate miR-27 degradation, we performed structurally informative mutagenic analyses of HSUR1. HSUR1 mutants in which the miR-27 binding site sequence is preserved, but sequestered in predicted helices, lose their ability to decrease miR-27 levels. These results indicate that the HSUR1 miR27-binding region must be available in a conformationally flexible segment for noncoding RNA function.

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The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3′ ends

Cited by 47 publications

References 56 publications

snRNA 3′ End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis

snRNA 3′ End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis

Methyltransferase-like protein 16 binds the 3′-terminal triple helix of MALAT1 long noncoding RNA

Host miRNA degradation by Herpesvirus saimiri small nuclear RNA requires an unstructured interacting region

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