Authors:	Kargapolova, Yulia
Title:	A novel crosslinking and immunoprecipitation method reveals the function of CSTF2tau in alternative processing of snRNAs
Online publication date:	15-Nov-2017
Year of first publication:	2017
Language:	english
Abstract:	RNA-binding proteins (RBPs) play a crucial role in the regulation of gene expression on various levels. RNA exists in a form of RNA-protein (RNP) particle throughout the lifespan. The composition and the structure of RNP vary between cell types, upon various stimuli and in time, and affect the fate of RNA. Mutations in RNA-binding proteins, therefore, can tremendously change the transcriptome profile of the cell and may be detrimental to the cell fate. Numerous mutations in RBPs are linked to inherited human diseases. The study of the mechanisms of recognition of target genes by RBPs and their role in the fate of targets is crucial for understanding disease mechanisms, treating and diagnostics of the human diseases. It may also have important implications for the future diagnostic and potential therapeutic strategies. The current study aims to improve currently existing approaches for illuminating the specificity of RNA-binding proteins (such as HITS-CLIP and iCLIP). The existing protocols are capable of detecting the RNA-protein interactions in a living cell. Yet they are characterized by complex biochemistry, usage of radioactivity, which is banned in many laboratories, and a protocol for library synthesis, which is based on an inherently low linker ligation efficiency. These limitations restrict the broad scientific community, especially nonexpert laboratories with bio-medical expertise in the usage of the protocols. To overcome the above-mentioned limitations, I modified the protocol. The modified protocol (conCLIP) is free of radioactivity, avoids using RNA-ligation on low input material, thus improving the complexity, robustness, and reproducibility of the cDNA synthesis procedure. The protocol also omits the size selection of cDNA, a step necessary to remove adaptor-adaptor contaminants and requires as little as 10 cycles of PCR amplification (which is at least 15 cycles less than in other protocols), thereby reducing non-desired amplification artifacts. I further designed a pipeline for the analysis of sequencing data, generated by conCLIP and confirmed the performance of both, the protocol and the pipeline by applying it to a known RNA-binding protein, CSTF2tau. The data, generated by conCLIP recapitulate previously described RNA recognition properties of this protein, it also reveals yet undescribed binding capacities. Particularly interesting properties of CSTF2tau, which have not been described before, are its binding to 5’ ends of replication-dependent histones as well as specific recognition of some small non-coding RNAs. My work also describes for the first time that small nuclear (sn)RNAs, involved in splicing, are significantly upregulated upon depletion of CSTF2tau. Interestingly, the same group of snRNAs is bound by the protein and the binding occurs at the 3’end of the molecules. I also reveal that a fraction of snRNAs is polyadenylated and this fraction decreases upon CSTF2tau depletion. Moreover, the depletion of the protein stabilizes snRNAs. I propose a model, which suggests a new mechanism by which the level of the snRNAs can be regulated via the activation of internal oligoadenylation sites. Upon depletion, internal polyadenylation sites become less efficient. In contrast, high-level CSTF2tau increases the usage of cryptic cleavage sites, which triggers oligoadenylation of snRNAs (presumably with other processing components) and results in their fast degradation.
DDC:	570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 04 Medizin FB 10 Biologie
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-1368
URN:	urn:nbn:de:hebis:77-diss-1000016519
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	https://rightsstatements.org/vocab/InC/1.0/
Extent:	118 Blätter
Appears in collections:	JGU-Publikationen