Small nucleolar RNAs (snoRNAs) function primarily as guide RNAs for posttranscriptional modification of rRNAs and spliceosomal snRNAs, both of which are functionally important and evolutionarily conserved molecules. It is commonly believed that snoRNAs and the modifications they mediate are highly conserved across species. However, most relevant data on snoRNA annotation and RNA modification are limited to studies on human and yeast. Here, we used RNA-sequencing data from the giant oocyte nucleus of the frog Xenopus tropicalis to annotate a nearly complete set of snoRNAs. We compared the frog data with snoRNA sets from human and other vertebrate genomes, including mammals, birds, reptiles, and fish. We identified many Xenopus-specific (or nonhuman) snoRNAs and Xenopus-specific domains in snoRNAs from conserved RNA families. We predicted that some of these nonhuman snoRNAs and domains mediate modifications at unexpected positions in rRNAs and snRNAs. These modifications were mapped as predicted when RNA modification assays were applied to RNA from nine vertebrate species: frogs X. tropicalis and X. laevis, newt Notophthalmus viridescens, axolotl Ambystoma mexicanum, whiptail lizard Aspidoscelis neomexicana, zebrafish Danio rerio, chicken, mouse, and human. This analysis revealed that only a subset of RNA modifications is evolutionarily conserved and that modification patterns may vary even between closely related species. We speculate that each functional domain in snoRNAs (half of an snoRNA) may evolve independently and shuffle between different snoRNAs.

Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB)-specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at similar to 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2'-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

Small nucleolar (sno)RNAs guide posttranscriptional modifications essential for the biogenesis and function of their target. The majority of snoRNAs in higher eukaryotes are encoded within introns. They are first released from nascent transcripts in the form of a lariat and rapidly targeted by the debranching enzyme and nuclear exonucleases for linearization and further trimming. In this study, we report that some snoRNAs are encoded within unusually stable intronic RNAs. These intronic sequences can escape the debranching enzyme and accumulate as lariats. Stable lariats bearing a snoRNA, or slb-snoRNA, are associated with snoRNA binding proteins but do not guide posttranscriptional modification. While most slb-snoRNAs accumulate in the nucleus, some can be exported to the cytoplasm. We find that this export competes with snoRNA maturation. Slb snoRNAs provide a previously unknown layer of regulation to snoRNA and snoRNA binding proteins.

In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified post-transcriptionally. Pseudouridylation and 2'-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-Psi 609, despite some noncanonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs. In Xenopus cells, a guide RNA generated from scaRNA, but not from snoRNA, could induce an additional pseudouridylation of U2 snRNA at position 60; both guide RNAs were equally active on a U2 snRNA-specific substrate in yeast cells. Thus, post-transcriptional modification of functionally important RNAs, such as rRNAs and snRNAs, is highly regulated and more complex than simply strong base-pairing between a guide RNA and substrate RNA. We discuss possible regulatory roles for these unexpected modifications.