Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern.

Clément Bouton,Maria Dimitrova,Angèle Geldreich,Lyubov A. Ryabova,Mario Keller,Laëtitia Ramel

doi:10.1371/journal.pone.0132665

Abstract

The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.

Highlights

Alternative RNA splicing is intensively performed by eukaryotes to increase the proteome diversity through the formation of numerous mRNA isoforms from a primary transcript and to regulate the expression of proteins in different organs and cell types [1,2,3]
Alternative splicing of 35S RNA potentially occurs in many Cauliflower mosaic virus (CaMV) isolates since they possess three of the four splice donor sites and the splice acceptor site described for Cabb-S isolate [18]
Analysis of amplified cDNAs obtained by RT-PCR of viral RNAs extracted from Cabb B-JIinfected turnip plants revealed that the splicing pattern of 35S RNA is more complex than previously described for Cabb-S isolate: we identified sixteen isoforms resulting from the use of six splice donor sites and seven acceptor sites

Summary

Introduction

Alternative RNA splicing is intensively performed by eukaryotes to increase the proteome diversity through the formation of numerous mRNA isoforms from a primary transcript and to regulate the expression of proteins in different organs and cell types [1,2,3]. Several plant DNA viruses from the Geminiviridae and Caulimoviridae families perform splicing to express some of their proteins [10,11]. In Mastreviruses (family Geminiviridae), splicing of the C transcript generates an mRNA that codes for the fusion protein Rep which is required to initiate DNA replication by rolling circle mechanism [10]. In the Caulimoviridae family, whose members are all pararetroviruses, splicing has only been described so far in two genera—Tungrovirus and Caulimovirus—and it has been proposed to enhance the expression of distal ORFs in the spliced RNAs. In Rice tungro bacilliform virus (RTBV), the polycistronic 35S RNA undergoes a single splicing event that leads to removal of a large intron (6.3 kb), resulting in a monocistronic mRNA specific for protein P4 [13]. The biological relevance of splicing in RTBV and FMV has never been investigated

Methods

Results

Conclusion