RNA Biology最新文献

Liver diseases are a significant global health issue, characterized by elevated levels of disorder and death. The substantial impact of ageing on liver diseases and their prognosis is evident. Multiple processes are involved in the ageing process, which ultimately leads to functional deterioration of this organ. The process of liver ageing not only renders the liver more susceptible to diseases but also compromises the integrity of other organs due to the liver's critical function in metabolism regulation. A growing body of research suggests that long non-coding RNAs (lncRNAs) play a significant role in the majority of pathophysiological pathways. They regulate gene expression through a variety of interactions with microRNAs (miRNAs), messenger RNAs (mRNAs), DNA, or proteins. LncRNAs exert a major influence on the progression of age-related liver diseases through the regulation of cell proliferation, necrosis, apoptosis, senescence, and metabolic reprogramming. A concise overview of the current understanding of lncRNAs and their potential impact on the development of age-related liver diseases will be provided in this mini-review.

We show that a small biotin-binding RNA aptamer that folds into a pseudoknot structure acts as a substrate for bacterial RNase P RNA (RPR) with and without the RNase P C5 protein. Cleavage in the single-stranded region in loop 1 was shown to depend on the presence of a RCCA-motif at the 3' end of the substrate. The nucleobase and the 2'hydroxyl at the position immediately 5' of the cleavage site contribute to both cleavage efficiency and site selection, where C at this position induces significant cleavage at an alternative site, one base upstream of the main cleavage site. The frequencies of cleavage at these two sites and Mg²⁺ binding change upon altering the structural topology in the vicinity of the cleavage site as well as by replacing Mg²⁺ with other divalent metal ions. Modelling studies of RPR in complex with the pseudoknot substrates suggest alternative structural topologies for cleavage at the main and the alternative site and a shift in positioning of Mg²⁺ that activates the H₂O nucleophile. Together, our data are consistent with a model where the organization of the active site structure and positioning of Mg²⁺ is influenced by the identities of residues at and in the vicinity of the site of cleavage.

tRNA nucleotidyltransferase represents a ubiquitous and essential activity that adds the indispensable CCA triplet to the 3'-end of tRNAs. To fulfill this function, the enzyme contains a set of highly conserved motifs whose coordinated interplay is crucial for the sequence-specific CCA polymerization. In the human enzyme, alterations within these regions have been shown to lead to the manifestation of disease. Recently, we developed an in vivo screening system that allows for the selection and analysis of tRNA nucleotidyltransferase variants by challenging terminal AMP incorporation into tRNA during induced RNase T-catalyzed CCA-decay. Here, we extend this method for screening of full CCA-end repair by utilizing the CCA-trimming activity of exonuclease LCCR4. To demonstrate the combined potential of these two in vivo selection systems, we applied a semi-rational library design to investigate the mode of operation of catalytically important motifs in the human CCA-adding enzyme. This approach revealed unexpected requirements for amino acid composition in two motifs and gives new insights into the mechanism of CCA addition. The data show the potential of these RNase-based screening systems, as they allow the detection of enzyme variations that would not have been identified by a conventional rational approach. Furthermore, the combination of both RNase T and LCCR4 systems can be used to investigate and dissect the effects of pathogenic mutations on C- and A-addition.

Mutations in PKD1 coding sequence and abnormal PKD1 expression levels contribute to the development of autosomal-dominant polycystic kidney disease, the most common genetic disorder. Regulation of PKD1 expression by factors located in the promoter and 3´ UTR have been extensively studied. Less is known about its regulation by 5´ UTR elements. In this study, we investigated the effects of uORFs and uORF-affecting variants by combining bioinformatic analyses, luciferase reporter assays, RT-qPCR and immunoblotting experiments. Our analyses demonstrate that PKD1 mRNA contains two evolutionarily conserved translation-inhibitory uORFs. uORF1 is translatable, and uORF2 is likely not translatable. The 5´ UTR and uORFs do not modulate downstream protein output under endoplasmic reticulum stress and oxidative stress conditions. Some of uORF-perturbing variants in the SNP database are predicted to affect gene translation. Luciferase reporter assays and RT-qPCR results reveal that rs2092942382 and rs1596636969 increase, while rs2092942900 decreases main gene translation without affecting transcription. Antisense oligos targeting the uORFs reduce luciferase protein levels without altering luciferase mRNA levels. Our results establish PKD1 as a novel target of uORF-mediated translational regulation and mutations that perturb uORFs may dysregulate PKD1 protein level.

Germ cells depend on specialized post-transcriptional regulation for proper development and function, much of which is mediated by dynamic RNA granules. These membrane-less organelles form through the condensation of RNA and proteins, governed by multivalent biomolecular interactions. RNA granules compartmentalize cellular components, selectively enriching specific factors and modulating biochemical reactions. Over recent decades, various types of RNA granules have been identified in germ cells across species, with extensive studies uncovering their molecular roles and developmental significance. This review explores the mRNA regulatory mechanisms mediated by RNA granules in germ cells. We discuss the distinct spatial organization of specific granule components and the variations in material states of germ granules, which contribute to the regulation of mRNA storage and translation. Additionally, we highlight emerging research on how changes in these material states, during developmental stages, reflect the dynamic nature of germ granules and their critical role in development.

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.