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

Abstract

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 ...

Cajal body function in genome organization and transcriptome diversity

“…This suggests CB regulation of snRNA production can alter competitive balances with other small RNAs to affect splicing. For example, a snoRNA was found that competes with U1 snRNA for a binding site in RNA, leading to mis-splicing for the E2F7 transcription factor [126]. As such, the CB is an important regulator of small RNA expression and function (Figures 2, 3 and 4).…”

Specialized box C/D snoRNPs act as antisense guides to target RNA base acetylation

“…These snoRNAs are classified as ‘orphan’ snoRNAs. Recent studies have shown that box C/D snoRNAs not only target rRNA and snRNA but also mRNAs and that they may have functions not related to site-directed methylation of ribose sugars [8–10]. In particular, fragments derived from snoRNAs (sdRNAs–snoRNAs derived) have been identified that exhibit miRNA-like characteristics and regulate alternative mRNA splicing in several species including mammals [9,11].…”

Rpl13a small nucleolar RNAs regulate systemic glucose metabolism

“…Although our study links the snoRNAs to altered mitochondrial function and ROS production, the precise molecular targets of these snoRNAs responsible for the observed physiological phenotypes remain to be determined. Beyond the predicted 28S and 18S ribosomal RNA targets for these snoRNAs (41), the Rpl13a snoRNAs may also function non-canonically to target mRNAs, given recent studies demonstrating that some snoRNAs target mRNAs (42,43) and our previous observation that the Rpl13a snoRNAs accumulate in the cytoplasm under metabolic stress conditions (12). Our RNAseq analysis suggests that rather than affecting the abundance of potential mRNA targets, the Rpl13a snoRNAs may impact their translation potential.…”