Study of the RNA splicing kinetics via in vivo 5-ethynyl uridine labelling.

Splicing, a process of intron removal from eukaryotic RNA transcripts, is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency giving rise to alternative splicing products. At the same time, splice sites might be utilised at a variable rate. We used 5-ethynyl uridine labelling to sequence a nascent transcriptome of HeLa cells and deduce the rate of splicing for each donor and acceptor splice site. The following correlation analysis allowed us to assess a correspondence of primary transcript features with the rate of splicing. Some dependencies we revealed were anticipated, such as splicing rate decrease with a decreased complementarity of donor splice site to U1 and acceptor sites to U2 snRNAs, or an acceleration of donor site usage if an upstream acceptor site is located at a shorter distance. Other dependencies were more surprising, like a negative influence of a distance to the 5' end on the rate of acceptor splicing site utilization, or the differences in splicing rate between long, short and RBM17-dependent introns. We also observed a deceleration of last intron splicing with an increase of the distance to the polyA site, which might be explained by a cooperativity of the splicing and polyadenylation. In addition, we performed the analysis of splicing kinetics of SF3B4 knockdown cells which suggested the impairment of U2 snRNA recognition step. As a result, we deconvoluted the effects of several examined features on the splicing rate into a single regression model. The data obtained here are useful for further studies in the field as it provides general splicing rate dependencies as well as helps justify the existence of slowly removed splice sites, e.g. to ensure alternative splicing.

Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.