High-throughput identification of C/D box snoRNA targets with CLIP and RiboMeth-seq

The ribose of RNA nucleotides can be 2′-O-methylated (Nm). Despite advances in high-throughput detection, the inert chemical nature of Nm still limits sensitivity and precludes mapping in mRNA. We leveraged the differential reactivity of 2′-O-methylated and 2′-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. Nm-seq uncovered thousands of Nm sites in human mRNA with features suggesting functional roles.

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“…In addition to these Nm sites, accumulating evidence suggests that internal positions in mRNA could also be 2′- O -methylated 20,21 .…”

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confidence: 99%

Nm-seq maps 2′-O-methylation sites in human mRNA with base precision

et al. 2017

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“…Most of these modifications are constitutive and play a role in rRNA folding and translational fidelity [25]. However, some rRNA sites are only partially methylated and some SNORDS exhibit sequence complementarity to rRNAs without a resulting modification, which suggests additional functions for SNORDs [26,27]. A few SNORDs including SNORD3@ and U8 and U13 direct pre-rRNA cleavage [28].…”

Section: The Classic Picture Of Snornasmentioning

confidence: 99%

C/D‐box snoRNAs form methylating and non‐methylating ribonucleoprotein complexes: Old dogs show new tricks

et al. 2017

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C/D box snoRNAs (SNORDs) are an abundantly expressed class of short, non-coding RNAs that have been long known to perform 2′-O-methylation of rRNAs. However, approximately half of human SNORDs have no predictable rRNA targets, and numerous SNORDs have been associated with diseases that show no defects in rRNAs, among them Prader-Willi syndrome, Duplication 15q syndrome and cancer. This apparent discrepancy has been addressed by recent studies showing that SNORDs can act to regulate pre-mRNA alternative splicing, mRNA abundance, activate enzymes, and be processed into shorter ncRNAs resembling miRNAs and piRNAs. Furthermore, recent biochemical studies have shown that a given SNORD can form both methylating and non-methylating ribonucleoprotein complexes, providing an indication of the likely physical basis for such diverse new functions. Thus, SNORDs are more structurally and functionally diverse than previously thought, and their role in gene expression is under-appreciated. The action of SNORDs in non-methylating complexes can be substituted with oligonucleotides, allowing devising therapies for diseases like Prader-Willi syndrome.

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“…For a listing of yeast snoRNA-target interactions see ( https://www-snorna.biotoul.fr/ ) ( Lestrade & Weber, 2006 ). In addition, a number of recent reports have described methods for the identification of RNA-RNA interactions through proximity ligation followed by sequencing of the products of reverse transcription and PCR amplification (RT-PCR) ( Gumienny et al , 2017 ; Kudla et al , 2011 ; Sharma et al , 2016 ; Sugimoto et al , 2015 ). In the crosslinking and sequencing of hybrids (CLASH) approach, stringent tandem affinity purification including denaturing conditions is used to recover only covalent RNA-protein interactions ( Kudla et al ., 2011 ).…”

Section: Introductionmentioning

confidence: 99%

Mapping targets for small nucleolar RNAs in yeast

Dudnakova¹,

Dunn-Davies²,

Peters³

et al. 2018

Wellcome Open Res

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Background: Recent analyses implicate changes in the expression of the box C/D class of small nucleolar RNAs (snoRNAs) in several human diseases. Methods: Here we report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH) with the snoRNP proteins Nop1, Nop56 and Nop58. We also developed a bioinformatics approach to filter snoRNA-target interactions for bona fide methylation guide interactions. Results: We recovered 241,420 hybrids, out of which 190,597 were classed as reproducible, high energy hybrids. As expected, the majority of snoRNA interactions were with the ribosomal RNAs (rRNAs). Following filtering, 117,047 reproducible hybrids included 51 of the 55 reported rRNA methylation sites. The majority of interactions at methylation sites were predicted to guide methylation. However, competing, potentially regulatory, binding was also identified. In marked contrast, following CLASH performed with the RNA helicase Mtr4 only 7% of snoRNA-rRNA interactions recovered were predicted to guide methylation. We propose that Mtr4 functions in dissociating inappropriate snoRNA-target interactions. Numerous snoRNA-snoRNA interactions were recovered, indicating potential cross regulation. The snoRNAs snR4 and snR45 were recently implicated in site-directed rRNA acetylation, and hybrids were identified adjacent to the acetylation sites. We also identified 1,368 reproducible snoRNA-mRNA interactions, representing 448 sites of interaction involving 39 snoRNAs and 382 mRNAs. Depletion of the snoRNAs U3, U14 or snR4 each altered the levels of numerous mRNAs. Targets identified by CLASH were over-represented among these species, but causality has yet to be established. Conclusions: Systematic mapping of snoRNA-target binding provides a catalogue of high-confidence binding sites and indicates numerous potential regulatory interactions.

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High-throughput identification of C/D box snoRNA targets with CLIP and RiboMeth-seq

Cited by 40 publications

References 71 publications

Nm-seq maps 2′-O-methylation sites in human mRNA with base precision

Nm-seq maps 2′-O-methylation sites in human mRNA with base precision

C/D‐box snoRNAs form methylating and non‐methylating ribonucleoprotein complexes: Old dogs show new tricks

Mapping targets for small nucleolar RNAs in yeast

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