A plethora of new functions of non-coding RNAs have been discovered in past few years. In fact, RNA is emerging as the central player in cellular regulation, taking on active roles in multiple regulatory layers from transcription, RNA maturation, and RNA modification to translational regulation. Nevertheless, very little is known about the evolution of this \Modern RNA World' and its components. In this contribution we attempt to provide at least a cursory overview of the diversity of non-coding RNAs and functional RNA motifs in non-translated regions of regular messenger RNAs (mRNAs) with an emphasis on evolutionary questions. This survey is complemented by an in-depth analysis of examples from different classes of RNAs focusing mostly on their evolution in the vertebrate lineage. We present a survey of Y RNA genes in vertebrates, studies of the molecular evolution of the U7 snRNA, the snoRNAs E1/U17, E2, and E3, the Y RNA family, the let-7 microRNA family, and the mRNA-like evf-1 gene. We furthermore discuss the statistical distribution of microRNAs in metazoans, which suggests an explosive increase in the microRNA repertoire in vertebrates. The analysis of the transcription of non-coding RNAs (ncRNAs) suggests that small RNAs in general are genetically mobile in the sense that their association with a hostgene (e.g. when transcribed from introns of a mRNA) can change on evolutionary time scales. The let-7 family demonstrates, that even the mode of transcription (as intron or as exon) can change among paralogous ncRNA.

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The spliceosome is one of the largest and most dynamic molecular machines in the cell. The central part of the complex is formed by five small nuclear ribonucleoproteins (snRNPs) which are generated in a multi-step biogenesis pathway. Moreover, the snRNPs undergo extensive rearrangements during the splicing and require reassembly after every intron removal. Both de novo assembly and post-splicing recycling of snRNPs are guided and facilitated by specific chaperones. Here, I reveal molecular details of function of two snRNP chaperones, SART3 and TSSC4. While TSSC4 is a previously uncharacterized protein, SART3 has been described before as a U6 snRNP-specific factor which assists in association of U6 and U4 particles into di-snRNP, and is important for the U4/U6 snRNP recycling. However, the mechanism of its function has been unclear. Here, I provide an evidence that SART3 interacts with a post-splicing complex and propose that SART3 could promote its disassembly. Our data further suggest that SART3 binds U6 snRNP already within the post-splicing complex and thus participates in the whole recycling phase of U6 snRNP. Then, I show that TSSC4 is a novel U5 snRNP-specific chaperone which promotes an assembly of U5 and U4/U6 snRNPs into a splicing-competent tri-snRNP particle. We identified...

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Long non-coding RNAs (lncRNAs) are key regulators of gene expression, playing crucial roles in biological processes across many species, tissues and diseases. The liver is a highly responsive organ in which large changes in gene expression are perpetuated by a myriad of internal and external stimuli; as such, the liver makes an ideal system in which to study lncRNAs. Global patterns of expression, maturation and localization were established for both lncRNA and protein-coding gene (PCG) transcripts across five subcellular compartments in male and female mouse liver, both with and without exposure to TCPOBOP, a direct agonist of the nuclear receptor CAR. In contrast to PCGs, lncRNAs showed very strong enrichment for tight chromatin binding, which increased the sensitivity for lncRNA detection and facilitated discovery of many novel sex-biased and xenobiotic-responsive lncRNAs. These findings helped identify candidate regulatory lncRNAs based on their co-localization within topologically associating domains, or their transcription divergent or antisense to PCGs associated with pathways linked to liver physiology and disease. The liver cell type-specific expression of lncRNAs and PCGs was assessed by single nucleus RNA-seq (snRNA-seq). Liver sexual dimorphism was largely restricted to hepatocyte populations, where many sex-biased genes exhibited zonated expression. Changes in lncRNA and PCG expression following exposure to endogenous hormones (growth hormone) and exogenous chemicals (TCPOBOP) was assessed, identifying cell cluster-specific perturbations to native sex-bias and hepatocyte zonation-dependent gene expression, and highlighting the interconnectedness between liver sexual dimorphism and zonation of the hepatic lobule at the single nuclei level. Finally, an in vivo method for epigenetic reprogramming of lncRNAs using a dual adeno-associated virus delivery system was utilized to knockdown two TCPOBOP-inducible lncRNAs in mouse liver. The knockdown phenotype of one of these lncRNAs, established by snRNA-seq, suggests it plays a functional role in regulating cholesterol metabolism and transport, triglyceride catabolism, and pyruvate metabolism in mouse liver. Together, these studies characterize hepatic lncRNA expression patterns, on both the sub-cellular and single cell levels, and present a strategy for interrogating the roles of specific lncRNAs in liver tissue in vivo. / 2023-01-18T00:00:00Z

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Messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II) and has to undergo multiple processing events before it can be translated into a protein: a cap structure is added to its 5’ end, noncoding, intervening sequences (introns) are removed and coding exons are ligated together and a poly(A) tail is added to its 3’end. Splicing, the process of intron removal, is carried out in the spliceosome, a megacomplex comprehending up to 300 proteins. The core components of the spliceosome that directly interact with the pre-mRNA are the small nuclear ribonucleoprotein particles (snRNPs). They consist of one of the U-rich snRNAs U1, U2, U4, U5 or U6 together with several particle-specific proteins and core proteins. All mRNA processing events can occur co-transcriptionally, i.e. while the RNA is still attached to the gene via Pol II. The in vivo studies of co-transcriptional RNA processing events had been possible only in special biological systems by immunoelectron microscopy and only recently, Chromatin Immunoprecipitation (ChIP) made it possible to investigate cotranscriptional splicing factor assembly on genes. My thesis work is divided into two parts: Part I shows that the core components of the splicing machinery are recruited co-transcriptionally to mammalian genes in vivo by ChIP. The co-transcriptional splicing factor recruitment is dependent on active transcription and the presence of introns in genes. Furthermore, a new assay was developed that allows for the first time the direct monitoring of co-transcriptional splicing in human cells. The topoisomerase I inhibitor camptothecin increases splicing factor accumulation on the c-fos gene as well as co-transcriptional splicing levels, which provides direct evidence that co-transcriptional splicing events depend on the kinetics of RNA synthesis. Part II of the thesis is aimed to investigate whether Pol II has a functional role in the biogenesis of the U6 snRNA, which is the RNA part of the U6 snRNP involved in splicing. Pol III had been shown to transcribe the U6 snRNA gene, but ChIP experiments revealed that Pol II is associated with all the active U6 snRNA gene promoters. Pol II inhibition studies uncovered that U6 snRNA expression and probably 3’end formation is dependent on Pol II.

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