The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element G / C GAC G / C and also 59 splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 59 end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 59 splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction.

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5 splice site, can positively influence utilization of an upstream 3 splice site via exon definition in both trans-and cis-splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5 splice site on trans-splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5 splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient transsplicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis-spliced intron. Weakening of the 3 splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between U1 and U2 snRNPs whether this interaction is across introns or exons.Pre-mRNA splicing is a process whereby introns are removed, and the exons are linked together to produce mature mRNA molecules. Central to this reaction are the small nuclear ribonucleoprotein particles (snRNPs) 1 including U1, U2, U4/ U6, and U5 in addition to a multitude of protein factors (1). Each of the snRNPs consists of a small RNA complexed with both common as well as specific proteins (2). Functions have been assigned to the snRNPs involved in splicing including the interaction of U1 with the 5Ј splice site, interaction of U2 snRNP with the branch site sequence, U5 interaction with the free 5Ј exon generated after the first step of the reaction, and contact between U6 and the 5Ј splice site region subsequent to the interaction with U1.The trans-splicing reaction occurring in a growing list of lower eukaryotes, including trypanosomatids (3, 4), nematodes (5), trematodes (6), euglenoids (7), ascidians (8), and cnidarians (9) is characterized by essential similarities as well as fundamental differences with standard cis-splicing of pre-mRNA introns (for reviews, see Refs. 10 and 11). The most striking feature of trans-splicing is the fact that the 5Ј exon is donated from a separate RNA, the spliced leader RNA (SL RNA) (12, 13). The SL RNA, when complexed with proteins in the functional SL RNP, has essential characteristics of the Sm snRNPs involved in numerous RNA-processing reactions (14 -16). In nematodes, these traits include a 5Ј trimethylguanosine cap, binding of the Sm core proteins, and the presence of snRNPspecific prote...