Functional characterization of C/D snoRNA-derived microRNAs

by Gustavo Nicolas Lemus Diaz
Doctoral thesis
Date of Examination:2017-12-08
Date of issue:2018-06-26
Advisor:Dr. Jens Gruber
Referee:Prof. Dr. Reinhard Lührmann
Referee:PD Dr. Halyna Shcherbata
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Steven Johnsen
Persistent Address: http://dx.doi.org/10.53846/goediss-6945

 

 

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Abstract

English

miRNAs are essential regulators of cell fate and involved in several human diseases, although their biogenesis is a well-accepted paradigm (microprocessor, Exp5, Dicer, and Argonaute). New evidence revealed different biogenesis for miRNAs distinct to canonical miRNA pathway; this new source of miRNAs including mirtrons, Exp1 dependent and Dicer-independent miRNAs, furthermore also others ncRNAs like tRNAs and snoRNAs produce fragments that reflects Dicer processing and Argonaute incorporation in sRNA profiles, however, functional evaluation and mechanisms are still poorly described. Studying C/D snoRNA-derived miRNAs using NGS profiles (sRNA-seq and CLIP of ribonucleoproteins) called small RNA fragments derived from C/D snoRNA loci poorly recovered from miRNP (Argonaute, Dicer) but strongly associated to snoRNPs (Nop56, Nop58, and FBL). However, U3 (C/D snoRNA) suited as a bona fide source of miRNAs including defined mRNA targets. To test functionality in-vivo at high resolution, I generated, implemented and validated a single cell assay for miRNA posttranscriptional regulation. Using this system U3 derived fragments perform as low expressed miRNAs in vivo in the Hek293 model and hold mRNA endogenous targets. In conclusion, using NGS data and the high-resolution reporter system, this study showed that non-canonical U3 C/D snoRNA is a bona fide miRNA source, while intronic C/D snoRNA-derived fragments rather might be degradation products.
Keywords: snoRNA; miRNA; NGS; Analytical flow cytometry; Genomic data science; bioinformatics; Dual reporter assays
 
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