RNA Biology最新文献

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2^Max, a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2^Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2^Max amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2^Max in cellular gene excision. In sum, CasX2^Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.

Viroids, small circular non-coding RNAs, act as infectious pathogens in higher plants, demonstrating high stability despite consisting solely of naked RNA. Their dependence of replication on host machinery poses the question of whether RNA modifications play a role in viroid biology. Here, we explore RNA modifications in the avocado sunblotch viroid (ASBVd) and the citrus exocortis viroid (CEVd), representative members of viroids replicating in chloroplasts and the nucleus, respectively, using LC - MS and Oxford Nanopore Technology (ONT) direct RNA sequencing. Although no modification was detected in ASBVd, CEVd contained approximately one m⁶A per RNA molecule. ONT sequencing predicted three m⁶A positions. Employing orthogonal SELECT method, we confirmed m⁶A in two positions A353 and A360, which are highly conserved among CEVd variants. These positions are located in the left terminal region of the CEVd rod-like structure where likely RNA Pol II and and TFIIIA-7ZF bind, thus suggesting potential biological role of methylation in viroid replication.

Long non-coding RNAs (lncRNAs) exert a significant influence on the occurrence and progression of osteoarthritis (OA). LncRNAs are characterized by their multifunctional nature, capable of regulating the expression, transcription, translation, and structural function of target genes through various mechanisms, spanning epigenetic, transcriptional, post-transcriptional, and post-translational levels. This review examines the mechanisms and functions of lncRNAs in cell proliferation, differentiation, apoptosis, extracellular matrix (ECM) degradation, and inflammatory responses in chondrocytes, synovial cells, and mesenchymal stem cells (MSCs) from mice and humans associated with OA. We emphasize the integral role of lncRNAs in the OA disease process. Conclusively, we present insights into OA treatment from the perspective of targeting lncRNAs, addressing future development prospects and potential clinical applications.

Alterations in cellular metabolism are known to play a crucial role in the development and progression of cancer. The lipoxygenase pathway, which controls unsaturated fatty acid metabolism, has been shown to regulate tumour progression and is commonly altered in breast cancer. In this study, we first obtained 11 lipoxygenase pathway genes from the Molecular Signatures Database (MSigDB). We then explored the lipoxygenase pathway-related long non-coding RNAs (lncRNAs) in breast cancer tissues from The Cancer Genome Atlas (TCGA) database. Information from our analysis was used to construct a risk prediction model (ProlncSig) to predict breast cancer prognosis. We found that ProlncSig could accurately identify breast cancer patients that had significantly shorter overall survival from those that had longer overall survival. Moreover, ProlncSig performs better in predicting prognosis than the clinicopathological features. Furthermore, GO and KEGG enrichment analysis showed that ProlncSig risk score had a high correlation with immune signature. We developed and validated an accurate prognostic risk prediction model (ProlncSig) based on lipoxygenase pathway-related lncRNAs, which has strong potential to provide prognostic information and may provide novel guidance for immunotherapeutic strategies for breast cancer patients.

Pre-mRNA splicing, carried out in the nucleus by a large ribonucleoprotein machine known as the spliceosome, is functionally and physically coupled to the mRNA surveillance pathway in the cytoplasm called nonsense-mediated mRNA decay (NMD). The NMD pathway monitors for premature translation termination, which can result from alternative splicing, by relying on the exon junction complex (EJC) deposited on exon-exon junctions by the spliceosome. Recently, multiple genetic screens in human cell lines have identified numerous spliceosome components as putative NMD factors. Using publicly available RNA-seq datasets from K562 and HepG2 cells depleted of 18 different spliceosome components, we found that natural NMD-targeted mRNA isoforms were upregulated when catalytic spliceosome members were reduced. While some of this increase could be due to widespread pleiotropic effects of spliceosome dysfunction (e.g. reduced expression of NMD factors due to missplicing of their mRNAs), we identified that AQR, SF3B1, SF3B4, and CDC40 may have a more direct role in NMD. We also tested the hypothesis that increased production of novel NMD substrates may overwhelm the pathway to find a direct correlation between the amount of novel NMD substrates detected and the degree of NMD inhibition observed. Finally, similar transcriptome alterations and NMD substrate upregulation were observed in cells treated with spliceosome inhibitors and in cells derived from retinitis pigmentosa patients with mutations in PRPF8 and PRPF31. Overall, our results show that regardless of the cause, spliceosome disruption upregulates a broad set of NMD targets, which could contribute to cellular dysfunction in spliceosomopathies.

MicroRNA-mediated gene silencing is a conserved mechanism of post-transcriptional gene regulation across metazoans. It depends on base pairing between small RNAs and mRNAs, and on protein complexes including the RNA-induced silencing complex (RISC), where Argonaute 2 (AGO2) plays a central role. A full understanding of RNA silencing requires reliable molecular tools to study AGO2 and RISC. Affinity tagging and antibody-based methods can introduce artefacts, and both the N- and C-terminal domains of AGO2 are critical for its function. While N-terminal tags are frequently used, and a recent study in mice showed altered activity in N-terminal HaloTag-AGO2 fusions, the consequences of C-terminal tagging remain underexplored. CRISPaint, a CRISPR-Cas9-based technique, enables endogenous C-terminal tag fusions without requiring homology arms. Using this system, we generated the first C-terminal HaloTag fusion of AGO2 (AGO2HALO) in human A549 cells. We found that the AGO2HALO fusion protein exhibits reduced binding with TNRC6A, with no effect on cell viability. However, it significantly impairs RNA cleavage, silencing activity, and nuclear localization. We further compared AGO2-EGFP and EGFP-AGO2 using transient transfection. N-terminally tagged AGO2 retained wild-type-like function and localization, while C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing, nuclear import, and P-body localization. These results demonstrate that a C-terminal HaloTag compromises AGO2 functionality and is unsuitable for studying RISC biology. Our findings highlight the importance of validating tagging strategies to avoid misleading conclusions due to tag-induced functional defects. Pre-print, bioRxiv.

Circular infectious RNAs have been known for several decades. Their biology has been intriguing from the beginning, partly due to the antithesis between their efficiency and tiny size. Amongst infectious circular RNAs viroids hold a special place not only because they were the first to be characterized as such but also because they have been extensively studied as a group. Viroids do not encode proteins and therefore have to rely for their biological cycle on the host factors. As a result, the identification and functional characterization of host factors enabling their biological cycle has been of prime importance to the community. With the advent of high throughput sequencing technologies, viroid-like infectious RNAs have been found in plants, fungi, and animals, including mammals, making understanding their biology even more interesting and important. In this review, we summarize what is known about the replication of these tiny yet very efficient infectious RNAs.

Accumulation of various genetics and epigenetics alterations are accepted to result in the initiation and progression of hepatocellular carcinoma (HCC), and its high metastasis is viewed as a critical bottleneck leading to its treatment failure. Amongst them, the microRNAs arising from the lack of the antioxidant transcription factor Nrf2 lead to cancer metastasis. However, much less is known about the regulation of microRNAs by Nrf1, even though it acts as an essential determinon of cell homoeostasis by governing the transcriptional expression of those driver genes contributing to the EMT involved in its metastasis. In this study, distinct EMT phenotypes resulted from specific knockouts of Nrf1 and Nrf2 in HepG2 cells, as accompanied by their differential migratory and invasive capabilities. The Nrf1α^-/--leading EMT results from a significant decrease in the epithelial CDH1 expression, plus another increased expression of the mesenchymal CDH2. Such distinct phenotypes of Nrf1α^-/- from Nrf2^-/- cell lines were also attributable to differential regulation of two key microRNAs, i.e. miR-3187-3p and miR-1247-5p. Further experiments also unravelled that Nrf1 activates the miR-3187-3p expression, directly targeting for the inhibition of SNAI1, leading to CDH1 activation but with CDH2 inhibition insomuch as to prevent the process of EMT. By contrast, Nrf2 inhibits the miR-1247-5p expression, relieving its inhibitory effect on MMP15 and MMP17 to promote the EMT. Collectively, these results demonstrate that the EMT of HCC is likely prevented by Nrf1 via the miR-3187-3p signalling to SNAI1-CDH1/2 axis, but conversely promoted by Nrf2 through the miR-1247-5p-MMP15/17 signalling axis.

Circular RNAs (circRNAs) are covalently closed single-stranded RNA molecules, which have been implicated in both physiology and human diseases. Most circRNAs are typically generated through backsplicing, where a downstream splice donor is covalently joined to an upstream splice acceptor. Backsplicing is dependent on the spliceosome machinery and is precisely controlled by various cis-elements and trans-factors. In the present review, we summarize the molecular mechanisms of backsplicing regulation as well as their physiological and pathological significance. Additionally, we discuss the strategies to manipulate circRNA expression in vivo and in vitro, aiming to explore the application of circRNA biogenesis in the diagnosis and therapy of human diseases.

Cellular differentiation requires highly coordinated action of all three transcriptional systems to produce rRNAs, mRNAs and various 'short' and 'long' non-coding RNAs by RNA Polymerase I, II and III systems, respectively. RNA Polymerase I catalyzes transcription of about 400 copies of mammalian rDNA genes, generating 18S, 5.8S and 28S rRNA molecules. Lens fiber cell differentiation is a unique process to study transcriptional mechanisms of individual crystallin genes as their very high transcriptional outputs are directly comparable only to globin genes in erythrocytes. Importantly, both terminally differentiated lens fiber cells and mammalian erythrocytes degrade their nuclei through different mechanisms. In lens, the generation of the organelle-free zone (OFZ) includes the degradation of mitochondria, endoplasmic reticulum, Golgi apparatus and nuclei. Here, using RNA fluorescence in situ hybridization (FISH), we evaluated nascent rRNA transcription, located in the nucleoli, during the process of mouse lens fiber cell differentiation. Lens fiber cell nuclei undergo morphological changes including chromatin condensation prior to their denucleation. Remarkably, nascent rRNA transcription persists in all nuclei that are in direct proximity of the OFZ. Additionally, changes in both nuclei and nucleoli shape were evaluated via immunofluorescence detection of fibrillarin, nucleolin, UBF and other proteins. These studies demonstrate for the first time that highly condensed lens fiber cell nuclei have the capacity to support nascent rRNA transcription. Thus, we propose that 'late' production of rRNA molecules and consequently of ribosomes increases crystallin protein synthesis machinery within the mature lens fibers.