Insights into the regulation of RNA helicases by protein cofactors

by Indira Memet
Doctoral thesis
Date of Examination:2019-02-05
Date of issue:2019-02-12
Advisor:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Peter Rehling
Referee:Dr. Ricarda Richter-Dennerlein
Referee:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Silvio Rizzoli
Persistent Address: http://dx.doi.org/10.53846/goediss-7279

 

 

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Abstract

English

RNA helicases comprise a large family of ubiquitously expressed enzymes that remodel RNA structures and RNA-protein complexes in an NTP-dependent manner. These proteins are essential regulators of every RNA-related process, including pre-mRNA splicing and ribosome biogenesis, where they are suggested to perform various activities, such as unwinding RNA duplexes and displacing proteins from RNA. RNA helicases require a complex regulation due to the lack of specificity of their conserved helicase core, their generally low intrinsic activity and the involvement of individual helicases in multiple cellular functions. The activity of these enzymes can be modulated in diverse ways, including through interactions with effector proteins termed helicase cofactors. Several helicase cofactors identified so far share a conserved glycine-rich domain known as a G-patch domain. In yeast, the role of these G-patch proteins as RNA helicase regulators has been established. However, a larger number of G-patch proteins are expressed in human cells and much less is known about their functions and interactions with RNA helicases. In this study, a comprehensive analysis of the human G-patch protein family was performed in order to assess their functions and potential role as cofactors of RNA helicases. Our results show that all 22 human G-patch proteins interact with an RNA helicase and, in most cases, they enhance the RNA binding affinity and/or the ATPase activity of their helicase partner. Only three human DEAH/RHA helicases associate with G-patch proteins, among which DHX15 is regulated by approximately 20 G-patch cofactors, suggesting that this helicase could have a central role in the cell. Our transcriptome analyses indicate that DHX15 and the majority of G-patch proteins are involved in alternative splicing, where they regulate specific genes but also have common targets. We confirm the role in alternative splicing for several proteins and propose that DHX15 functions together with its G-patch cofactors in this pathway. Our results further suggest that G-patch proteins and DHX15 could potentially regulate alternative splicing by direct binding to pre-mRNAs as well as in an indirect manner. In addition to its role in splicing, DHX15 also functions in ribosome biogenesis together with its G-patch cofactor NKRF and with the exonuclease XRN2. These proteins associate into a nucleolar complex that is involved in the efficient processing of the pre-rRNA transcript at a specific site. The catalytic activity of DHX15 is required for this function, implying that the helicase might remodel structures in this region to facilitate the cleavage event. Taken together, our data provide essential insights into the role of human G-patch proteins as cofactors of RNA helicases and also reveal functions for DHX15 in alternative splicing and ribosome biogenesis together with its G-patch cofactors. Therefore, the findings of this study provide the basis for further understanding the function and regulation of RNA helicases.
Keywords: RNA helicase; helicase cofactor
 
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