LINE-1 mRNA 3' end dynamics shape its biology and retrotransposition potential.

LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two ORFs, of which L1-ORF2p nicks genomic DNA and reverse transcribes L1 mRNA using the nicked DNA as a primer which base-pairs with poly(A) tail of L1 mRNA. To better understand the importance of non-templated L1 3' ends' dynamics and the interplay between L1 3' and 5' ends, we investigated the effects of genomic knock-outs and temporal knock-downs of XRN1, DCP2, and other factors. We hypothesized that in the absence of XRN1, the major 5'→3' exoribonuclease, there would be more L1 mRNA and retrotransposition. Conversely, we observed that loss of XRN1 decreased L1 retrotransposition. This occurred despite slight stabilization of L1 mRNA, but with decreased L1 RNP formation. Similarly, loss of DCP2, the catalytic subunit of the decapping complex, lowered retrotransposition despite increased steady-state levels of L1 proteins. In both XRN1 and DCP2 depletions we observed shortening of L1 3' poly(A) tails and their increased uridylation by TUT4/7. We explain the observed reduction of L1 retrotransposition by the changed qualities of non-templated L1 mRNA 3' ends demonstrating the important role of L1 3' end dynamics in L1 biology.

3D Puzzle at the Nanoscale-How do RNA Viruses Self-Assemble their Capsids into Perfectly Ordered Structures.

The phenomenon of RNA virus self-organization, first observed in the mid-20th century in tobacco mosaic virus, is the subject of extensive research. Efforts to comprehend this process intensify due to its potential for producing vaccines or antiviral compounds as well as nanocarriers and nanotemplates. However, direct observation of the self-assembly is hindered by its prevalence within infected host cells. One of the approaches involves in vitro and in silico research using model viruses featuring a ssRNA(+) genome enclosed within a capsid made up of a single type protein. While various pathways are proposed based on these studies, their relevance in vivo remains uncertain. On the other hand, the development of advanced microscopic methods provide insights into the events within living cells, where following viral infection, specialized compartments form to facilitate the creation of nascent virions. Intriguingly, a growing body of evidence indicates that the primary function of packaging signals in viral RNA is to effectively initiate the virion self-assembly. This is in contrast to earlier opinions suggesting a role in marking RNA for encapsidation. Another noteworthy observation is that many viruses undergo self-assembly within membraneless liquid organelles, which are specifically induced by viral proteins.

Applications of the versatile CRISPR-Cas13 RNA targeting system.

CRISPR-Cas are adaptable natural prokaryotic defense systems that act against invading viruses and plasmids. Among the six currently known major CRISPR-Cas types, the type VI CRISPR-Cas13 is the only one known to exclusively bind and cleave foreign RNA. Within the last couple of years, this system has been adapted to serve numerous, and sometimes not obvious, applications, including some that might be developed as effective molecular therapies. Indeed, Cas13 has been adapted to kill antibiotic-resistant bacteria. In a cell-free environment, Cas13 has been used in the development of highly specific, sensitive, multiplexing-capable, and field-adaptable detection tools. Importantly, Cas13 can be reprogrammed and applied to eukaryotes to either combat pathogenic RNA viruses or in the regulation of gene expression, facilitating the knockdown of mRNA, circular RNA, and noncoding RNA. Furthermore, Cas13 has been harnessed for in vivo RNA modifications including programmable regulation of alternative splicing, A-to-I and C to U editing, and m6A modifications. Finally, approaches allowing for the detection and characterization of RNA-interacting proteins have also been demonstrated. Here, we provide a comprehensive overview of the applications utilizing CRISPR-Cas13 that illustrate its versatility. We also discuss the most important limitations of the CRISPR-Cas13-based technologies, and controversies regarding them. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.