MicroRNA biogenesis is broadly disrupted by inhibition of the splicing factor SF3B1.

In animals, microRNA (miRNA) biogenesis begins with cotranscriptional cleavage of the primary (pri-)miRNA by the Microprocessor complex. Cotranscriptional splicing has been shown to influence Microprocessor cleavage when miRNAs are hosted in introns of protein-coding pri-miRNAs, but the impact of splicing on production of miRNAs hosted in long non-coding (lnc)RNAs is largely unknown. Here, we investigated the role of splicing in the biogenesis of miR-122, an lncRNA-hosted, highly expressed, medically important, liver-specific miRNA. We found that splicing inhibition by the SF3B1 inhibitor pladienolide B (PlaB) led to strong and rapid reduction in transcription of endogenous, but not plasmid-encoded, pri-miR-122, resulting in reduced production of mature miR-122. To allow detection of rapid changes in miRNA biogenesis despite the high stability of mature miRNAs, we used SLAMseq to globally quantify the effects of short-term splicing inhibition on miRNA synthesis. We observed an overall decrease in biogenesis of mature miRNAs following PlaB treatment. Surprisingly, miRNAs hosted in exons and introns were similarly affected. Together, this study provides new insights into the emerging role of splicing in transcription, demonstrating novel biological importance in promotion of miR-122 biogenesis from an lncRNA, and shows that SF3B1 is important for global miRNA biogenesis.

Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively.

Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing.

Comparative effects of viral-transport-medium heat inactivation upon downstream SARS-CoV-2 detection in patient samples.

Introduction. The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.Hypothesis. Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.Aim. We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).Methodology. We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.Results. Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically (n=127; R2=0.9259).Conclusion. Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.

Posttranscriptional Regulation of 14q32 MicroRNAs by the CIRBP and HADHB during Vascular Regeneration after Ischemia.

After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the microRNA genes' order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32, and we aimed to identify these RBPs. To identify proteins responsible for this posttranscriptional regulation, we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of cold-inducible RBP (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) to the precursors of late-upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hindlimb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329 but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB-/- 3T3 cells, which display reduced expression of miR-329 and miR-495 but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs.

Oleic Acid Induces MiR-7 Processing through Remodeling of Pri-MiR-7/Protein Complex.

MicroRNAs (miRs) play a vital role in governing cell function, with their levels tightly controlled at transcriptional and post-transcriptional levels. Different sets of RNA-binding proteins interact with primary miRs (pri-miRs) and precursor-miR transcripts (pre-miRs), controlling their biogenesis post-transcriptionally. The Hu antigen R (HuR)-mediated binding of Musashi homolog2 (MSI2) to the conserved terminal loop of pri-miR-7 regulates the levels of brain-enriched miR-7 formation in a tissue-specific manner. Here, we show that oleic acid (OA) inhibits the binding of proteins containing RNA recognition motifs (RRM) to the conserved terminal loop of pri-miR-7. Using electrophoretic mobility shift assays in HeLa cell extracts, we show that OA treatment disrupts pre-miR/protein complexes. Furthermore, OA rescues in vitro processing of pri-miR-7, which is otherwise blocked by HuR and MSI2 proteins. On the contrary, pri-miR-16 shows reduced processing in the presence of OA. This indicates that OA may inhibit the binding of other RRM-containing protein/s necessary for miR-16 processing. Finally, we demonstrate that OA induces mature miR-7 production in HeLa cells. Together, our results demonstrate that OA can regulate the processing of pri-miRs by remodeling their protein complexes. This provides a new tool to study RNA processing and a potential lead for small molecules that target the miR-7 biogenesis pathway.