RNA Biology最新文献

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.

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 interactions 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 during ribosomal subunit biogenesis. 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 interactions. 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.

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.

Fusion transcripts (FTs) are RNA molecules, also known as chimeric transcripts, formed through chromosomal rearrangements or transcriptional processes, contributing to tumorigenesis. This study systematically examined tumour-specific FTs in hepatocellular carcinoma (HCC) using high-throughput RNA sequencing data from independent datasets and The Cancer Genome Atlas (TCGA). Our meta cohort analysis included 328 HCC samples. Using STAR-Fusion, we identified 15 novel tumour-specific FTs, with SERPINA1-H19 as the most recurrent fusion event. Comparative expression analysis of fusion partner genes revealed significant downregulation in HCC tumours relative to normal adjacent liver tissues (NAT). We validated the expression levels of the key partner genes with 436 TCGA samples serving as an in silico validation cohort and in wet lab validation cohorts with 42 samples. ALB, APOA2, IGF2, MT2A, SERPINA1, and H19, which are key liver-associated genes, were frequently involved in tumour-specific fusion events suggesting either a loss of tumour suppressor property or gaining a novel function playing a role in hepatocarcinogenesis. Detailed characterization of SERPINA1-H19 identified 16 transcript variants with distinct structural modifications that may impact its functional output. Furthermore, low expression of SERPINA1 and H19 was associated with more aggressive HCC phenotypes. Overall, this study established a comprehensive repository of FTs for the first time, offering valuable insights into their role in HCC and their potential to serve as diagnostic and prognostic biomarkers for HCC.

R2 elements serve as a class of non-long terminal repeat (non-LTR) retrotransposons found in animal genomes that specifically insert into the ribosomal DNA (rDNA) sequences of host genomes. Each R2 element contains a single open reading frame (ORF) encoding a multifunctional protein with nucleic acid-binding, reverse transcriptase, and endonuclease activities, enabling specific genomic integration via a mechanism called target-primed reverse transcription (TPRT). As a classical model for studying retrotransposition mechanisms, R2 elements possess unique biological properties and precise integration capabilities, which have inspired novel genome engineering strategies. In this review, we summarize the components and integration mechanisms of R2 retrotransposons and highlight the recent advances in employing these mobile elements for targeted gene integration. Finally, we present future directions for the utilization of R2 retrotransposons as novel biotechnological tools.

Alternative splicing is a very important mechanism to diversify an organism's transcriptome with minimal increases in genome size. It can modify the function of the finished protein or affect its regulation, e.g. induce nonsense-mediated decay (NMD) to degrade the transcript. Mechanisms that affect alternative splicing are therefore of great interest. It has been shown that splicing can be affected by RNA secondary structures within pre-mRNAs. These structured regions of RNA (strucRNA) would affect their transcript in cis, but only a few such cases are known in plants. In this study, we interrogate plant genomes for cis-regulatory strucRNAs. By applying a comparative-genomics-based approach to 130 plant genomes, we identified 16 strucRNA candidates. Five candidates likely regulate in cis using alternative splicing and NMD. Other predictions might not regulate alternative splicing, including four putative small nucleolar RNAs (snoRNAs). Of our five cis-regulatory strucRNAs that are implicated in alternative splicing control, two are now experimentally validated in follow-up studies. These results stand in contrast to the few previously validated examples. Although we were able to predict some strucRNAs, all motifs had generally modest levels of covariation, which is a pattern of mutations that indicates a conserved secondary structure. With few mutations, comparative-genomics-based approaches to find strucRNAs are less effective. Other approaches of finding regulatory RNAs in plants might thus be needed, and more available genomic or transcriptomic data might improve the quality and quantity of promising candidates.

RNA-focused therapy and diagnostics have been making waves in molecular biology due to the advantages RNA has over DNA; for instance, the ability of RNA to target nearly any genetic component in the cell is a big step in treating disorders. Moreover, RNA-based diagnosis of diseases is only becoming increasingly popular, especially after the COVID-19 pandemic, which brought up the need for cost-effective and efficient diagnosing kits for the vast majority. RNA-based techniques also have close to no risk of genotoxicity and can efficiently target undruggable regions of the cell. RNA treatments have effectively shown the future of the medical industry in the past couple of decades, and they will only be seen to improve. This review paper provides an overview on the different techniques that use RNA-based approaches in the field of diagnostics and therapeutics.

Mesenchymal Stromal/Stem Cells (MSCs) are among the most frequently studied cell types in clinical trials, and their small extracellular vesicles (sEVs) are now being extensively investigated for therapeutic applications. The RNA cargo of MSC-sEVs, particularly miRNAs and mRNAs, is widely believed to be a key therapeutic component of these vesicles. In this review, we critically examine using first principles and peer-reviewed literature, whether MSC- extracellular vesicles (MSC-EVs) can deliver sufficient quantity of functional miRNA or mRNA to target compartments within recipient cells to elicit a pharmacological response. Several RNA sequencing studies reveal that miRNAs are underrepresented in the small RNA population of MSC-sEVs compared to the parent MSCs. Additionally, the majority of miRNAs are mature forms that are not associated with Argonaute (AGO) proteins, essential for their function in RNA-induced silencing complexes (RISCs). Compounding this, cellular uptake of EVs is generally inefficient, with less than 1% being internalized, and only a fraction of these reaching the cytosol. This suggests that EVs may not deliver miRNAs in sufficient quantities to meaningfully interact with AGO proteins, either through canonical or non-canonical pathways, or with other proteins like Toll-like receptors (TLRs). Further, MSC-sEV RNAs are generally small, with sizes less than 500 nucleotides indicating that any mRNA present is likely fragmented as the average mammalian mRNA is approximately 2000 nucleotides, a fact confirmed by RNA sequencing data. Together, these findings challenge the notion that RNA, particularly miRNAs and mRNAs, are primary therapeutic attributes of MSC-sEVs.

mRNAs undergo a series of chemical modifications to become competent for nuclear export and translation. This is referred to as mRNA maturation or processing and includes capping, splicing, and 3'end formation. These steps can be hijacked in cancer to alter proteins' forms and levels in the absence of mutation or changes to transcript levels. Here, we focus on an emerging idea that some factors act in multiple processing events and that their dysregulation in both their canonical and noncanonical functions contributes to cancer with a focus on Acute Myeloid Leukaemia (AML). As examples, we discuss the eukaryotic translation initiation factor (eIF4E), splice factor 3 complex B subunit 1 (SF3B1), U2 small nuclear auxiliary factor (U2AF1), and associated factors. These physically interact with each other and play roles in splicing, export, and translation. Malignant dysregulation of this mRNA processing-export-translation axis diversifies the proteome to support cancer. Finally, we discuss the simultaneous dysregulation of mRNA processing in malignancy and related therapeutic development.