Characterization of the Molecular Basis of Group II Intron RNA Recognition by CRS1-CRM Domains

Ayenachew Bezawork-Geleta,Felix Shaya,Max Kolton,Liron Klipcan,Oren Ostersetzer-Biran,Ido Keren

doi:10.1074/jbc.m710488200

Ayenachew Bezawork-Geleta, Felix Shaya + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m710488200

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Abstract

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Highlights

Teins, including the archaeal Alba protein, DNase I, the ribosomal protein S8, RNA 3Ј-terminal phosphate cyclase, C-terminal domain of prolyl-tRNA synthetase, and the THUMP domain, have been included into a protein family, known as the Alba superfamily (8)
Binding affinity of CRM1 to atpF intron RNA (ϳ65 nM), its Individual CRM Domains from CRS1 Bind with Different Affinities and Specificities to Different Fragments of the atpF Intron—Individual CRS1-CRM domains were expressed and purified as recombinant proteins fused to Glutathione S-Transferase (GST), and each protein was assayed for its binding to atpF intron RNA in vitro
We assayed the binding specificities associated with each of the CRM domains of CRS1 to different fragments of atpF intron RNA (Fig. 2A); these included a unique 111-nt sequence found within atpF domain I and domain IV, two previously identified CRS1binding sites (12), and several other fragments that are not recognized by the intact CRS1 protein, including atpF domains II, III, and V-VI and the plastid psaI mRNA (12)

Summary

Introduction

Teins, including the archaeal Alba protein, DNase I, the ribosomal protein S8, RNA 3Ј-terminal phosphate cyclase, C-terminal domain of prolyl-tRNA synthetase, and the THUMP domain, have been included into a protein family, known as the Alba superfamily (8). CRM3 demonstrated high affinity binding to RNA but lacked sequence specificity, CRM1 formed specific interactions with a unique region within domains I, the 111-nt sequence, of the atpF intron ligand of the intact CRS1 protein.

Results

Conclusion