‘Black sheep’ that don’t leave the double-stranded RNA-binding domain fold

Gleghorn, Michael L.; Maquat, Lynne E.

doi:10.1016/j.tibs.2014.05.003

Cited by 58 publications

(69 citation statements)

References 101 publications

(181 reference statements)

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“…Proteins recognize their RNA targets in a sequence-and/or a structuredependent manner, including single-, double-, or even fourstranded RNAs (10,11,17). In this work, we discovered that METTL16 interacts with the MALAT1 triple helix in vitro (Figs.…”

Section: Discussionmentioning

confidence: 84%

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.

220

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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Section: Discussionmentioning

confidence: 84%

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.

220

205

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“…1 A and B and Fig. S1) (42)(43)(44)(45). To verify the impairment of miR9897-3p-mediated silencing in dus16-1, we measured the abundance of the intact gluc Chlamydomonas expressing DUS16-Venus in a dus16-1 background (DUS16-Venus, strain #1 and #2) and wild-type cells expressing Venus (Venus, strain #3 and #4) were examined by confocal microscopy.…”

Section: Dus16 Ismentioning

confidence: 99%

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

Yamasaki

Onishi

Kim

et al. 2016

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

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Canonical microRNAs (miRNAs) are embedded in duplexed stemloops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii. In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM-or dsRBDtruncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.Chlamydomonas | microRNA biogenesis | dsRNA-binding protein | small RNA-seq | mutagenesis

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“…1A). 4,12 The second dsRBD of XlrbpA forms the prototype of canonical dsRBDs with the 2 a-helices packed against the 3-stranded anti-parallel b-sheet. 13,14 In addition, structures of dsRBDs in complex with dsRNA substrates revealed the canonical mode of RNA recognition by dsRBDs.…”

Section: Primary Function Of Dsrbdsmentioning

confidence: 99%

“…12,22,23 An interesting example of a type-B dsRBD involved in localization is the C-terminal dsRBD of human Dicer. Although processing of Dicer's substrates occurs in the cytoplasm of mammalian cells, several reports suggest that Dicer may also have additional roles in the nucleus.…”

Section: Dsrbds and Nuclear Importmentioning

confidence: 99%

Functions of double-stranded RNA-binding domains in nucleocytoplasmic transport

Banerjee

2014

RNA Biology

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International audienceThe double-stranded RNA-binding domain (dsRBD) is a small protein domain found in eukaryotic, prokaryotic and viral proteins, whose central property is to bind to double-stranded RNA (dsRNA). Aside from this major function, recent examples of dsRBDs involved in the regulation of the sub-cellular localization of proteins, suggest that the participation of dsRBDs in nucleocytoplasmic trafficking is likely to represent a widespread auxiliary function of this type of RNA-binding domain. Overall, dsRBDs from proteins involved in many different biological processes have been reported to be implicated in nuclear import and export, as well as cytoplasmic, nuclear and nucleolar retention. Interestingly, the function of dsRBDs in nucleocytoplasmic trafficking is often regulated by their dsRNA-binding capacity, which can either enhance or impair the transport from one compartment to another. Here, we present and discuss the emerging function of dsRBDs in nucleocytoplasmic transport

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‘Black sheep’ that don’t leave the double-stranded RNA-binding domain fold

Cited by 58 publications

References 101 publications

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

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

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

Functions of double-stranded RNA-binding domains in nucleocytoplasmic transport

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