We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18:1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.The splicing together of exons during the maturation of mRNA from a primary transcript represents a step that is fundamental in the transfer of information from DNA to protein for most genes in higher eukaryotes. This process is catalyzed by a supramolecular particle known as the spliceosome, which serves as the site for the two transesterification reactions that remove an intron and connect two adjacent exons. The spliceosome is composed of five small nuclear RNA molecules and perhaps hundreds of proteins (20,23,33,55). Despite this complexity, much has been learned about the identity of these components, their function, and the sequence of events that culminates in exon joining (2,5,16,21). Less understood are the earliest events that initiate splicing, in which the boundaries of the intron that must be removed are identified. The absolute accuracy of this molecular recognition is critical to gene function and must depend on signals comprised of sequence and/or structural elements in the RNA substrate.In higher eukaryotes, several classes of splicing sequence elements can be distinguished based on their function and location; the splice sites themselves constitute one such class (31, 39). The 5Ј splice site is comprised of a 9-base consensus sequence that includes an almost universally conserved GU surrounded by additional nucleotides that are less well conserved. Similarly, the 3Ј splice contains a highly conserved AG surrounded by a less conserved C and G and an upstream tract of about 10 bases that is rich in pyrimidines. Splice site sequences from tens of thousands of introns have been used to define these two consensus sequences and to construct position-specific scoring matrices to evalu...

RAG1 binding to TCR gene elements is dictated by transcriptional control elements and by transcription itself; these findings provide direct confirmation of the long-held accessibility model.

bAt the Tcrb locus, V␤-to-J␤ rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the "beyond 12/23" rule (B12/23 rule). Previous work showed that V␤ recombination signal sequences (RSSs) do not recombine with J␤ RSSs because J␤ RSSs are crippled for either nicking or synapsis. This result raised the following question: how can crippled J␤ RSSs recombine with D␤ RSSs? We report here that the nicking of some J␤ RSSs can be substantially stimulated by synapsis with a 3=D␤1 partner RSS. This result helps to reconcile disagreement in the field regarding the impact of synapsis on nicking. Furthermore, our data allow for the classification of Tcrb RSSs into two major categories: those that nick quickly and those that nick slowly in the absence of a partner. Slow-nicking RSSs can be stimulated to nick more efficiently upon synapsis with an appropriate B12/23 partner, and our data unexpectedly suggest that fast-nicking RSSs can be inhibited for nicking upon synapsis with an inappropriate partner. These observations indicate that the RAG proteins exert fine control over every step of V(D)J cleavage and support the hypothesis that initial RAG binding can occur on RSSs with either 12-or 23-bp spacers (12-or 23-RSSs, respectively).T he first step in the assembly and diversification of antigen receptor genes is V(D)J recombination, a site-specific recombination reaction that joins variable (V), diversity (D), and joining (J) coding segments at immunoglobulin and T-cell receptor (TCR) loci. During B-and T-cell development, the recombination-activating gene 1 and 2 proteins (RAG1/2) initiate V(D)J recombination (1, 2) at sites specified by recombination signal sequences (RSSs), which flank each V, D, and J gene segment.The biochemistry of V(D)J recombination can be divided into two phases: cleavage and joining (3, 4). The cleavage phase is catalyzed by a recombinase complex comprised of RAG1/2 (collectively, RAG) and the ubiquitous DNA binding/bending protein HMGB1 or the closely related HMGB2 protein (5). The first step in the cleavage phase is the binding of the recombinase complex to one RSS, forming a signal complex (SC). After SC formation, synapsis occurs, in which a partner RSS is captured to form the paired complex (PC) (6, 7). RAG catalyzes the nicking of DNA at the heptamer-RSS boundary, giving rise to a free hydroxyl group. Double-strand breaks are catalyzed in the PC via a transesterification reaction whereby the free hydroxyl created by nicking directly attacks the opposite strand (8). These four ends created by cleavage are resolved in the joining phase by the proteins of the nonhomologous end-joining (NHEJ) repair pathway to form a precise signal joint containing the RSSs and an imprecise coding joint containing the antigen receptor gene segments (reviewed in reference 9).Each RSS is comprised of three sequence elements: a relatively well-conserved heptamer (consensus CACAGTG) and nonamer (consensus ACAAAAACC) and a less well-conserved spacer of either 12 or 23 bp (12-RS...