Are Argonaute-Associated Tiny RNAs Junk, Inferior miRNAs, or a New Type of Functional RNAs?

The biosynthesis pathways of microRNAs (miRNAs) have been well characterized with the identification of the required components. miRNAs are synthesized from the transcripts of miRNA genes and other RNAs, such as introns, transfer RNAs, ribosomal RNAs, small nucleolar RNAs, and even viral miRNAs. These small RNAs are loaded into Argonaute (AGO) proteins and recruit the effector complexes to target mRNAs, repressing their gene expression post-transcriptionally. While mature miRNAs were defined as 19–23 nucleotides (nt), tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind AGOs as equivalent or lesser miRNAs compared to their full-length mature miRNAs. In contrast, my recent study revealed that when human AGO3 loads 14 nt cleavage-inducing tyRNAs (cityRNAs), comprised of the first 14 nt of their corresponding mature miRNA, it can become a comparable slicer to AGO2. This observation raises the possibility that tyRNAs play distinct roles from their mature form. This minireview focuses on human AGO-associated tyRNAs shorter than 19 nt and discusses their possible biosynthesis pathways and physiological benefits, including how tyRNAs could avoid target-directed miRNA degradation accompanied by AGO polyubiquitination.

Genome-Wide Identification and Analysis of Small Nucleolar RNAs and Their Roles in Regulating Latex Regeneration in the Rubber Tree (Hevea brasiliensis)

Small nucleolar RNAs (snoRNAs) are a class of conserved nuclear RNAs that play important roles in the modification of ribosomal RNAs (rRNAs) in plants. In rubber trees, rRNAs are run off with latex flow during tapping and need to be regenerated for maintaining the functions of the laticifer cells. SnoRNAs are expected to play essential roles in the regeneration of rRNAs. However, snoRNAs in the rubber tree have not been sufficiently characterized thus far. In this study, we performed nuclear RNA sequencing (RNA-seq) to identify snoRNAs globally and investigate their roles in latex regeneration. We identified a total of 3,626 snoRNAs by computational prediction with nuclear RNA-seq data. Among these snoRNAs, 50 were highly expressed in latex; furthermore, the results of reverse transcription polymerase chain reaction (RT-PCR) showed the abundant expression of 31 of these snoRNAs in latex. The correlation between snoRNA expression and adjusted total solid content (TSC/C) identified 13 positively yield-correlated snoRNAs. To improve the understanding of latex regeneration in rubber trees, we developed a novel insulated tapping system (ITS), which only measures the latex regenerated in specific laticifers. Using this system, a laticifer-abundant snoRNA, HbsnoR28, was found to be highly correlated with latex regeneration. To the best of our knowledge, this is the first report to globally identify snoRNAs that might be involved in latex regeneration regulation and provide new clues for unraveling the mechanisms underlying the regulation of latex regeneration.

The Arabidopsis 2′-O-Ribose-Methylation and Pseudouridylation Landscape of rRNA in Comparison to Human and Yeast

Eukaryotic ribosome assembly starts in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into the 35S pre-ribosomal RNA (pre-rRNA). More than two-hundred ribosome biogenesis factors (RBFs) and more than two-hundred small nucleolar RNAs (snoRNA) catalyze the processing, folding and modification of the rRNA in Arabidopsis thaliana. The initial pre-ribosomal 90S complex is formed already during transcription by association of ribosomal proteins (RPs) and RBFs. In addition, small nucleolar ribonucleoprotein particles (snoRNPs) composed of snoRNAs and RBFs catalyze the two major rRNA modification types, 2′-O-ribose-methylation and pseudouridylation. Besides these two modifications, rRNAs can also undergo base methylations and acetylation. However, the latter two modifications have not yet been systematically explored in plants. The snoRNAs of these snoRNPs serve as targeting factors to direct modifications to specific rRNA regions by antisense elements. Today, hundreds of different sites of modifications in the rRNA have been described for eukaryotic ribosomes in general. While our understanding of the general process of ribosome biogenesis has advanced rapidly, the diversities appearing during plant ribosome biogenesis is beginning to emerge. Today, more than two-hundred RBFs were identified by bioinformatics or biochemical approaches, including several plant specific factors. Similarly, more than two hundred snoRNA were predicted based on RNA sequencing experiments. Here, we discuss the predicted and verified rRNA modification sites and the corresponding identified snoRNAs on the example of the model plant Arabidopsis thaliana. Our summary uncovers the plant modification sites in comparison to the human and yeast modification sites.

Systematic mapping of small nucleolar RNA targets in human cells

Altered expression of box C/D small nucleolar RNAs (snoRNAs) is implicated in human diseases, including cancer. Box C/D snoRNAs canonically direct site-specific, 2′-O-methylation but the extent to which they participate in other functions remains unclear. To identify RNA targets of box C/D snoRNAs in human cells, we applied two techniques based on UV crosslinking, proximity ligation and sequencing of RNA hybrids (CLASH and FLASH). These identified hundreds of novel snoRNA interactions with rRNA, snoRNAs and mRNAs. We developed an informatic pipeline to rigorously call interactions predicted to direct methylation. Multiple snoRNA-rRNA interactions identified were not predicted to direct RNA methylation. These potentially modulate methylation efficiency and/or contribute to folding dynamics. snoRNA-mRNA hybrids included 1,300 interactions between 117 snoRNA families and 940 mRNAs. Human U3 is substantially more abundant than other snoRNAs and represented about 50% of snoRNA-mRNA hybrids. The distribution of U3 interactions across mRNAs also differed from other snoRNAs. Following U3 depletion, mRNAs showing altered abundance were strongly enriched for U3 CLASH targets. Most human snoRNAs are excised from pre-mRNA introns. Enrichment for snoRNA association with branch point regions of introns that contain snoRNA genes was common, suggesting widespread regulation of snoRNA maturation.

Emerging Data on the Diversity of Molecular Mechanisms Involving C/D snoRNAs

Box C/D small nucleolar RNAs (C/D snoRNAs) represent an ancient family of small non-coding RNAs that are classically viewed as housekeeping guides for the 2′-O-methylation of ribosomal RNA in Archaea and Eukaryotes. However, an extensive set of studies now argues that they are involved in mechanisms that go well beyond this function. Here, we present these pieces of evidence in light of the current comprehension of the molecular mechanisms that control C/D snoRNA expression and function. From this inventory emerges that an accurate description of these activities at a molecular level is required to let the snoRNA field enter in a second age of maturity.

NOP10 predicts lung cancer prognosis and its associated small nucleolar RNAs drive proliferation and migration

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide underlining the urgent need for new biomarkers and therapeutic targets for this disease. Long noncoding RNAs are critical players in NSCLC but the role of small RNA species is not well understood. In the present study, we investigated the role of H/ACA box small nucleolar RNAs (snoRNAs) and snoRNA-bound ribonucleoproteins (snoRNPs) in the tumorigenesis of NSCLC. H/ACA box snoRNPs including the NOP10 core protein were highly expressed in NSCLC. High levels of either NOP10 mRNA or protein were associated with poor prognosis in NSCLC patients. Loss of NOP10 and subsequent reduction of H/ACA box snoRNAs and rRNA pseudouridylation inhibited lung cancer cell growth, colony formation, migration, and invasion. A focused CRISPR/Cas9 snoRNA knockout screen revealed that genomic deletion of SNORA65, SNORA7A, and SNORA7B reduced proliferation of lung cancer cells. In line, high levels of SNORA65, SNORA7A, and SNORA7B were observed in primary lung cancer specimens with associated changes in rRNA pseudouridylation. Knockdown of either SNORA65 or SNORA7A/B inhibited growth and colony formation of NSCLC cell lines. Our data indicate that specific H/ACA box snoRNAs and snoRNA-associated proteins such as NOP10 have an oncogenic role in NSCLC providing new potential biomarkers and therapeutic targets for the disease.

Are spliced ncRNA host genes distinct classes of lncRNAs?

Many small nucleolar RNAs and many of the hairpin precursors of miRNAs are processed from long non-protein-coding host genes. In contrast to their highly conserved and heavily structured payload, the host genes feature poorly conserved sequences. Nevertheless, there is mounting evidence that the host genes have biological functions beyond their primary task of carrying a ncRNA as payload. So far, no connections between the function of the host genes and the function of their payloads have been reported. Here we investigate whether there is evidence for an association of host gene function or mechanisms with the type of payload. To assess this hypothesis we test whether the miRNA host genes (MIRHGs), snoRNA host genes (SNHGs), and other lncRNA host genes can be distinguished based on sequence and/or structure features unrelated to their payload. A positive answer would imply a functional and mechanistic correlation between host genes and their payload, provided the classification does not depend on the presence and type of the payload. A negative answer would indicate that to the extent that secondary functions are acquired, they are not strongly constrained by the prior, primary function of the payload. We find that the three classes can be distinguished reliably when the classifier is allowed to extract features from the payloads. They become virtually indistinguishable, however, as soon as only sequence and structure of parts of the host gene distal from the snoRNAs or miRNA payload is used for classification. This indicates that the functions of MIRHGs and SNHGs are largely independent of the functions of their payloads. Furthermore, there is no evidence that the MIRHGs and SNHGs form coherent classes of long non-coding RNAs distinguished by features other than their payloads.

Abstract 15984: Rpl13a Small Nucleolar RNAs Promote Oxidative Stress and Atherosclerosis

Reactive oxygen species (ROS) contribute to atherogenesis. An unusual mechanism that increases cellular ROS levels and oxidative stress involves 4 ubiquitously expressed noncoding small nucleolar RNAs (snoRNAs) from introns of the ribosomal protein L13a ( Rpl13a ) locus: U32a , U33 , U34 , and U35a . We tested the hypothesis that these snoRNAs promote aortic smooth muscle cell (SMC) activation and vascular inflammation, by using “snoKO” mice with targeted deletion of the 4 snoRNAs (but not Rpl13a ). Compared with congenic WT SMCs, snoKO SMCs showed 40±20% lower ROS levels, assessed by DCF fluorescence ( p <0.02). Congruently, ROS levels were 35±5% lower in snoKO than WT aorta and carotid frozen sections ( p <0.01), assessed by CellROX Orange fluorescence. Proliferation and migration evoked by FBS and PDGF-BB, respectively, were each 30±10% less in snoKO than WT SMCs ( p <0.01 for each). To assess SMC migration and proliferation in vivo, we performed carotid artery endothelial denudation. Before injury, snoKO and WT carotid arteries were morphologically equivalent. Four wk after injury, carotid neointimal hyperplasia was 57±9% less and luminal area was 40±20 % more in snoKO than in WT mice ( p <0.01). WT and snoKO mice had equivalent heart rates and systolic blood pressures by tail-cuff plethysmography: 480±20 vs 420±80 beats/min; 133±5, 132±7 mm Hg, respectively (n=5/group). To test whether snoRNAs affect atherosclerosis, we orthotopically transplanted carotid arteries from WT and snoKO mice into congenic Apoe -/- mice. Six wk post-op, atherosclerotic neointima was 70±10% smaller in snoKO than in WT carotids ( p <0.01). To assess SMC-to-foam-cell transdifferentiation, which is ROS-dependent, carotid cross-sections were stained for apoE to identify graft-derived cells and for cholesteryl ester with BODIPY. BODIPY + foam cells comprised 21±3% and 11±7% of neointimal area in WT and snoKO carotids, respectively ( p <0.05). Confocal co-localization of apoE and BODIPY (optical slice thickness 1 μm) showed that graft-derived foam cells were 2.0±0.6-fold more prevalent in WT than in snoKO carotids ( p <0.01). We conclude that Rpl13a snoRNAs promote SMC ROS levels, proliferation and migration in vitro and in vivo, and that these snoRNAs augment atherosclerosis.