Mapping 2′-O-Methyl Groups in Ribosomal RNA

Maden, B.E.H.

doi:10.1006/meth.2001.1250

Cited by 97 publications

(94 citation statements)

References 32 publications

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“…These two sites within U81 snoRNA optimally fulfill the requirements for efficient cleavage according to siRNA target-design rules (Elbashir et al 2002). In addition, the cleavage site at position 32 is located within the D9 antisense element of U81 snoRNA, which has been reported to guide methylation of A 391 within 28S rRNA (Maden 2001), and thus, is likely to represent one of the most accessible sites within U81 snoRNA.…”

Section: Resultsmentioning

confidence: 97%

Methodological obstacles in knocking down small noncoding RNAs

Ploner¹,

Ploner²,

Lukasser³

et al. 2009

RNA

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In the recent past, several thousand noncoding RNA (ncRNA) genes have been predicted within eukaryal genomes. However, for their functional analysis only a few high-throughput methods are currently available to knock down selected ncRNA species, such as microRNAs, which are targeted by antisense probes, termed antagomirs. We thus compared the efficiencies of four knockdown strategies, previously mainly employed for the analysis of protein-coding genes, to study the function of ncRNAs, in particular, small nucleolar RNAs (snoRNAs). Thereby, the class of snoRNAs represents one of the most abundant ncRNA species. The majority of snoRNAs has been shown to mediate nucleotide modifications by targeting ribosomal RNAs (rRNAs) through complementary antisense elements. However, some snoRNAs, termed ''orphan snoRNAs,'' lack telltale complementarities to rRNAs and thus their function remains elusive. We therefore applied RNA interference (RNAi), locked nucleic acid (LNA), or peptide nucleic acid antisense approaches, as well as a ribozyme-based strategy to knock down a snoRNA. As a proof of principle, we targeted the canonical U81 snoRNA, which has been shown to mediate modification of nucleotide A 391 within eukaryal 28S rRNA. Our results demonstrate that while RNAi is an unsuitable tool for snoRNA knockdown, a ribozyme-based strategy, as well as an LNA-antisense oligonucleotide approach, resulted in a decrease of U81 snoRNA expression levels up to 60%. However, no concomitant decrease in enzymatic activity of U81 snoRNA was observed, indicating that improvement of more efficient knockdown techniques for ncRNAs will be required in the future.

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Section: Resultsmentioning

confidence: 97%

Methodological obstacles in knocking down small noncoding RNAs

Ploner¹,

Ploner²,

Lukasser³

et al. 2009

RNA

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“…5D). Although in some instances, this method fails to detect a 2′-O-methylation (55), the absence of a reverse transcription stop together with fibrillarin not being detected in nuclear supernatant strongly suggest that E2F7 pre-mRNA is not methylated.…”

Section: Snord27mentioning

confidence: 96%

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Falaleeva

Pagès

Matuszek

et al. 2016

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

165

152

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C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2′-Omethylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex, SNORD27 regulates the alternative splicing of the transcription factor E2F7 pre-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes.alternative splicing | gene regulation | snoRNAs | pre-mRNA processing S mall nucleolar RNAs (snoRNAs) are 60-to 300-nt-long noncoding RNAs that accumulate in the nucleolus. Based on conserved sequence elements, snoRNAs are classified as C/D box small nucleolar RNAs (SNORDs) or H/ACA box snoRNAs (SNORAs). SNORDs contain sequence elements termed C (RUGAUGA) and D (CUGA) boxes, usually present in duplicates (C′ and D′ boxes), and up to two antisense boxes that hybridize to the target RNA (1). In humans, SNORDs are usually derived from introns. After the splicing reaction, introns are excised as lariats, which are then opened by the debranching enzyme and subsequently degraded. Intronic SNORDs escape this degradation by forming a protein complex that consists of non-histone chromosome protein 2-like 1 (NHP2L1, 15.5K, SNU13), nucleolar protein 5A (NOP56), nucleolar protein 5 (NOP58), and fibrillarin (2-4). The SNORD protein complex forms through the entry of the snoRNA and fibrillarin to a complex containing NHP2L1, NOP58, and at least five assembly factors (5). The SNORD acts as a scaffold for the final protein complex formation and also controls recognition of other RNAs using the antisense boxes. The antisense boxes recognize sequences in rRNA, resulting in the fifth nucleotide upstream of the D or D′ box being 2′-O-methylated by fibrillarin (1). Structural studies indicate that the active form of SNORDs is dimeric (6).The conserved overall structure of SNORDs allows the identification of their putative target RNA binding sites. However, numerous SNORDs without obvious target RNAs have been identified (7-10) and are termed "orphan snoRNAs." Genome-wide deep sequencing experiments identified shorter but stable SNORD fragments that were found in all species tested, ranging from mammals to the protozoan Giardia lamblia (11) and EpsteinBarr virus (12). Fragments longer than 27 nt generated by SNORDs will ...

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“…2e). At lower concentration of dNTPs, reverse transcriptase halts 1 nt before the modification sites (Maden 2001). The appearance of a stop after incorporation of a nucleotide opposite template (U4) nucleotide A67 in native but not synthetic, unmodified U4 RNA strongly suggests that A66 on U4 snRNA is modified in vivo (Fig.…”

Section: Methylation Gguide (C/d Box) Rnasmentioning

confidence: 98%

“…For sequencing reaction, 1 mg of in vitro transcribed RNA was used with 1 mM each of ddNTPS. The positions of 29-O-methyls were identified by primer extension in the presence of limiting amount of dNTPs (0.01 mM) (Maden 2001).…”

Section: Mapping 29-o-methyl Groupsmentioning

confidence: 99%

Structural RNAs of known and unknown function identified in malaria parasites by comparative genomics and RNA analysis

Chakrabarti¹,

Pearson²,

Grate³

et al. 2007

RNA

115

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As the genomes of more eukaryotic pathogens are sequenced, understanding how molecular differences between parasite and host might be exploited to provide new therapies has become a major focus. Central to cell function are RNA-containing complexes involved in gene expression, such as the ribosome, the spliceosome, snoRNAs, RNase P, and telomerase, among others. In this article we identify by comparative genomics and validate by RNA analysis numerous previously unknown structural RNAs encoded by the Plasmodium falciparum genome, including the telomerase RNA, U3, 31 snoRNAs, as well as previously predicted spliceosomal snRNAs, SRP RNA, MRP RNA, and RNAse P RNA. Furthermore, we identify six new RNA coding genes of unknown function. To investigate the relationships of the RNA coding genes to other genomic features in related parasites, we developed a genome browser for P. falciparum (http://areslab.ucsc.edu/cgi-bin/hgGateway). Additional experiments provide evidence supporting the prediction that snoRNAs guide methylation of a specific position on U4 snRNA, as well as predicting an snRNA promoter element particular to Plasmodium sp. These findings should allow detailed structural comparisons between the RNA components of the gene expression machinery of the parasite and its vertebrate hosts.

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Mapping 2′-O-Methyl Groups in Ribosomal RNA

Cited by 97 publications

References 32 publications

Methodological obstacles in knocking down small noncoding RNAs

Methodological obstacles in knocking down small noncoding RNAs

Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing

Structural RNAs of known and unknown function identified in malaria parasites by comparative genomics and RNA analysis

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