The YTH Domain Is a Novel RNA Binding Domain

Zhaiyi Zhang,Dominik Theler,Katarzyna H Kaminska,Michael Hiller,Pierre De La Grange,Rainer Pudimat,Ilona Rafalska,Bettina Heinrich,Janusz M Bujnicki,Frédéric H.-T Allain,Stefan Stamm

doi:10.1074/jbc.m110.104711

Abstract

The YTH (YT521-B homology) domain was identified by sequence comparison and is found in 174 different proteins expressed in eukaryotes. It is characterized by 14 invariant residues within an alpha-helix/beta-sheet structure. Here we show that the YTH domain is a novel RNA binding domain that binds to a short, degenerated, single-stranded RNA sequence motif. The presence of the binding motif in alternative exons is necessary for YT521-B to directly influence splice site selection in vivo. Array analyses demonstrate that YT521-B predominantly regulates vertebrate-specific exons. An NMR titration experiment identified the binding surface for single-stranded RNA on the YTH domain. Structural analyses indicate that the YTH domain is related to the pseudouridine synthase and archaeosine transglycosylase (PUA) domain. Our data show that the YTH domain conveys RNA binding ability to a new class of proteins that are found in all eukaryotic organisms.

Highlights

The binding of proteins to RNA is a fundamental aspect of biology that interferes with most aspects of gene expression and cellular functions
We show that the YTH domain is a novel RNA binding domain that binds to a short, degenerated, single-stranded RNA sequence motif
The conservation of aromatic residues within the ␤ stands of the YTH domain is reminiscent of the RNA recognition motif (RRM), pseudouridine synthase and archaeosine transglycosylase (PUA), and OB-fold structures

Summary

EXPERIMENTAL PROCEDURES

Recombinant YT521-B—Recombinant YT521-B was generated by cloning full-length YT521-B [7] and a YTH domain deletion mutant (YTHdel) into the vector pFastBac HTa (Invitrogen). Minigene Analysis—Minigene analysis was essentially performed as described [16]. SiRNA Analysis of Minigenes—siRNA analysis of minigenes was performed in HEK293 cells. On the day of transfection, 100 ng of minigenes and 34.5 ng of siRNA were diluted in 25 ␮l of HBS buffer (20 mM HEPES and 150 mM NaCl, pH 7.4). 42 h after transfection cells were harvested for RNA and protein isolation. Expression, and Purification of the YTH Domain for NMR Study—The YTH domain (amino acids 346 –502) of Rattus norvegicus YT521-B protein was cloned into pTYB11 vector (New England Biolabs). This vector was transformed into Escherichia coli BL21(DE3) Codon ϩRIL (Stratagene). Figures of the NMR structure of the YTH domain were prepared using the program MOLMOL [20]

RESULTS

The YTH Binding Motif Causes

DISCUSSION