Interactions and functions of RNA-binding proteins
by Jens Kretschmer
Date of Examination:2017-01-20
Date of issue:2018-04-13
Advisor:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Heinz Neumann
Referee:Prof. Dr. Claudia Höbartner
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Jörg Stülke
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Abstract
English
Alongside several well-known modifications in DNA and proteins, more than 100 different types of chemical modification are also found in cellular RNAs. RNA modifications can influence the secondary structure and interactions of the RNAs that carry them and they can therefore play important roles in regulating the functions of the RNAs. For many RNA modifications, the enzymes that introduce them are known but the modification targets of several predicted modification enzymes remain to be identified. Interestingly, a particular modification, N6-methyladenosine (m6A), was recently found to be reversible and a group of proteins, termed “readers” that can recognise the modification often via a specialised RNA binding domain (YTH domain), have been identified. Such “reader” proteins have been shown to regulate the fate of RNAs according to their modification status, suggesting that this so called “epitranscriptome” is an additional layer of regulation of gene expression. In this study, cross-linking and analysis of cDNA (CRAC) was used to identify the RNAinteractome of the five human YTH domain-containing proteins, YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2. To facilitate the mapping of the deep sequencing data obtained from CRAC experiments performed in human cells, a bioinformatic pipeline was adapted and further developed. Analysis of the CRAC data showed that YTHDF1, YTHDF2, YTHDF3 and YTHDC1 predominantly cross-link to mRNAs, which is in line with recently published reports describing functions for these proteins in mRNA degradation, alternative pre-mRNA splicing and enhancing mRNA translation. Interestingly, the CRAC analysis of YTHDC2 revealed a specific cross-linking site on the 18S ribosomal RNA and the association of this protein with ribosomal complexes was confirmed by independent experimental approaches. CRAC analysis using truncated versions of YTHDC2 suggested that the R3H RNA binding domain is required for stable association of this protein with the ribosome and in vitro anisotropy experiments demonstrated that the YTH domain of YTHDC2 has a higher affinity for m6A modifications present in the sequence context found in the ribosomal RNAs than the classical m6A consensus motif found in many mRNAs. Interestingly, immunoprecipitation experiments followed by mass spectrometry identified the cytoplasmic 5’-3’ exonuclease XRN1 as an interaction partner of YTHDC2. These data could suggest a model in which recognition of the m6A modification(s) on the ribosomal RNA by the YTH domain of YTHDC2 promotes RNA degradation by XRN1. Taken together, this study contributes to the understanding of the diverse functions that modification “reader” proteins can play in regulating RNA metabolism.
Keywords: RNA biology; RNA modification; ribosome