Influence of RNA Secondary Structure on the Pre-mRNA Splicing Process

Abstract

Pre-mRNA splicing in eukaryotes requires joining together the nucleotides of the various mRNA-coding regions (exons) after recognizing them from the normally vastly superior number of non-mRNA-coding sequences (introns). For three excellent reviews on general splicing and its regulation, refer to references 14, 62, and 70. In eukaryotes, the vast majority of splicing processes are catalyzed by the spliceosome, a very complex RNA-protein aggregate which has been estimated to contain several hundred different proteins in addition to five spliceosomal snRNAs (1,54,62,63,81,109). These factors are responsible for the accurate positioning of the spliceosome on the 5Ј and 3Ј splice site sequences. The reason why so many factors are needed reflects the observation that exon recognition can be affected by many pre-mRNA features such as exon length (5, 97), the presence of enhancer and silencer elements (8, 62), the strength of splicing signals (45), the promoter architecture (29,55), and the rate of RNA processivity (86). In addition, the general cellular environment also exerts an effect, as recent observations suggest the existence of extensive coupling between splicing and many other gene expression steps (69) and even its modification by external stimuli (96).In the midst of all this complexity, it has also been proposed that pre-mRNA secondary structures can potentially influence splicing activity. However, despite a steady increase of reports invoking their effects on splicing regulation, the last specific review on this subject is now more than 10 years old (3). Here, we propose to address again this specific issue in the current perspective of the general field. Before we do this, however, we have to answer a basic question. DO PRE-mRNAS PRESENT SECONDARY STRUCTURE IN VIVO?Two properties of RNA molecules cannot be denied: their natural tendency to form highly stable secondary and tertiary structures in vitro and in vivo (9, 27, 39) and the observation that alterations in these structures represent a well-known regulatory mechanism for many RNA cellular processes (60).In this particular respect, however, a question that still remains to be addressed conclusively regards the presence of secondary structures in pre-mRNAs in vivo. That this existence may not simply be taken for granted comes from early experimental evidence. In fact, it was suggested that in vitro evidence regarding the possible influence of RNA structure on splicing (94) could not be accurately reproduced in vivo (95). The reason why this should be so goes back to the classical concept that RNA is coated in vivo by proteins. In fact, heterogeneous ribonucleoprotein particles have been known since early studies and the major protein family involved, the hnRNP proteins, are very abundant in mammalian cells. These RNAprotein interactions may well prevent mRNAs from folding in stable secondary structures (34) (Fig. 1a). For this reason, it was hypothesized that, following transcription, pre-mRNA may be allowed only a very limited timespan to fold (36)....

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