Artificial Box C/D RNAs Affect Pre-mRNA Maturation in Human Cells

Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2’-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2’-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2’-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2’-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2’-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2’-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans.

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“…The analogues of box C/D RNAs were obtained via in vitro transcription according to [ 18 , 19 ]: 28A4518 (98 nt)…”

Section: Methodsmentioning

confidence: 99%

Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues

et al. 2015

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“…For example, the neuron-specific SNORD115 promotes the inclusion of an alternative exon in the serotonin receptor 2C pre-mRNA (31), and SNORD88C regulates alternative splicing of FGFR3 pre-mRNA (32). Although proof of principle experiments have shown that methylation of the branch point adenosine by engineered snoRNAs can change alternative splicing (33)(34)(35), the mechanism by which SNORDs regulate splice site selection in vivo is not clear.…”

mentioning

confidence: 99%

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.

171

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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“…Antisense elements to the branch point, splice donor and acceptor sites, as well as the first and last nucleotides of the intron all resulted in increased alternative splicing and increased production of a known minor isoform of HSPA8 . In a follow‐up study, a subset of these artificial U24 snoRNAs directed at HSPA8 pre‐mRNA were also shown to cause a significant decrease in mRNA levels . As for the snoMEN constructs, this knockdown effect was dependent on the complementary interaction between snoRNA and target, but independent of the ability of the snoRNA to guide methylation of the target …”

Section: Noncanonical Targets and Functions For Snornasmentioning

confidence: 96%

“…In a follow‐up study, a subset of these artificial U24 snoRNAs directed at HSPA8 pre‐mRNA were also shown to cause a significant decrease in mRNA levels . As for the snoMEN constructs, this knockdown effect was dependent on the complementary interaction between snoRNA and target, but independent of the ability of the snoRNA to guide methylation of the target …”

Section: Noncanonical Targets and Functions For Snornasmentioning

confidence: 99%

The emerging landscape of small nucleolar RNAs in cell biology

2015

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Small nucleolar RNAs (snoRNAs) are a large class of small noncoding RNAs present in all eukaryotes sequenced thus far. As a family, they have been well characterized as playing a central role in ribosome biogenesis, guiding either the sequence-specific chemical modification of pre-rRNA (ribosomal RNA) or its processing. However, in higher eukaryotes, numerous orphan snoRNAs were described over a decade ago, with no known target or ascribed function, suggesting the possibility of alternative cellular functionality. In recent years, thanks in great part to advances in sequencing methodologies, we have seen many examples of the diversity that exists in the snoRNA family on multiple levels. In this review, we discuss the identification of novel snoRNA members, of unexpected binding partners, as well as the clarification and extension of the snoRNA target space and the characterization of diverse new noncanonical functions, painting a new and extended picture of the snoRNA landscape. Under the deluge of novel features and functions that have recently come to light, snoRNAs emerge as a central, dynamic, and highly versatile group of small regulatory RNAs. WIREs RNA 2015, 6:381–397. doi: 10.1002/wrna.1284

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Artificial Box C/D RNAs Affect Pre-mRNA Maturation in Human Cells

Cited by 10 publications

References 37 publications

Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues

Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues

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

The emerging landscape of small nucleolar RNAs in cell biology

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