RNA Folding Affects the Recruitment of SR Proteins by Mouse and Human Polypurinic Enhancer Elements in the Fibronectin EDA Exon

Abstract

In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]).While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing.The splicing process is a very flexible and critical step in gene expression. In fact, selected removal or inclusion of individual exons from nascent mRNA molecules allows a single gene to generate multiple proteins with different primary structures; in these cases, the process is known as alternative splicing (1, 38). Constitutive and alternative splicing processes are catalyzed by the spliceosome, a very complex RNA-protein aggregate that has been recently estimated to contain approximately 145 different proteins in addition to the five spliceosomal snRNAs (1,38,41,68). These factors are responsible for accurate positioning of the spliceosome on the 5Ј and 3Ј splice sequences that define the exon. However, correct positioning of the spliceosome is a very complex process owing to the degeneracy of the splice site consensus sequences, the presence of cryptic splice sites in large introns, and the fact that most pre-mRNAs contain multiple introns (26). Therefore, the action of several different proteins is required to achieve accurate positioning of the spliceosome on the splice site. Not surprisingly, alterations in the splicing process have been increasingly reported as being involved in many genetic diseases (5,8,13,23,58). Among the well-known factors that may heavily influence the identification of intron-exon boundaries by the spliceosome are the exon length (3, 65), the presence of splicing enhancer and silencer elements (5, 38), the strength of splicing signals (26), and the promoter architecture (19,33). In addition to these factors, it has been proposed that the natural tendency of RNAs to fold in highly stable secondary and tertia...

TDP-43 Binds Heterogeneous Nuclear Ribonucleoprotein A/B through Its C-terminal Tail

“…RNA Secondary Structure Determination-RNA secondary structure determination using limited V1 RNase, T1 RNase, and S1 nuclease digestion has been described in detail (45).…”

“…The probed RNA thus comprised the entire CFTR exon 9 sequence carrying a (ug) 11 u 7 repeat and a significant portion of the intervening sequence 8 intron. Structural probing was performed according to a methodology recently described for the EDA exons of the mouse fibronectin gene (45), which integrates limited RNase digestion with mFold structural predictions (46). As shown in Fig.…”

Repair of pre-mRNA splicing

“…27,28 However, it is clear that splicing defects may also arise by mutations that cause steric block of SS, 30 modulate SS communication 31,32 or change RNA folding. 33,34 It is interesting that only very few mutations have been found in the core spliceosomal components and the splicing regulatory proteins, certainly due to lethal effects during early development. 35 An increasing number of mutations that is being recognized are those that affect regulatory sites in RNA (binding sites for SR and hnRNP proteins and other splicing regulators).…”

Synonymous mutations in CFTR exon 12 affect splicing and are not neutral in evolution

“…In addition, exonic splicing elements that interact with several classes of positive and negative trans-acting splicing regulatory factors contribute significantly to constitutive and alternative splicing regulation (3,17). The sequence composition of exonic splicing regulatory sequences is highly degenerated (3,(18)(19)(20)(21), and their effect on splicing may be significantly affected by the context (13,14) and͞or depend on the formation of RNA secondary structures (22,23). The application of computational strategies indicates that exonic splicing regulatory sequences may be remarkably common in the exons of higher eukaryotes (20,24,25).…”