Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5' splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5' part of a beta-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (> 70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently cross-linked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5' part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.

Mutually exclusive splicing of exons 6A and 6B from the chicken ␤-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them. Splicing is the process by which introns from premessenger RNAs are removed in eukaryotes. Pre-mRNA splicing takes place within the spliceosome, which is a large, highly dynamic complex composed of four small ribonucleoprotein particles (snRNP 1 U1, U2, U4/U6, and U5) and multiple non-snRNP factors (1, 2). One of the most intriguing questions that remains in RNA splicing is how the 5Ј and 3Ј splice sites are selected and paired together within large RNA sequences (3). This question takes on particular importance in alternative splicing, where the selection of certain splice sites is modulated depending on the developmental stage, on tissue differentiation, or on metabolic changes of the cells (3). Numerous studies have demonstrated that regulatory sequences within the pre-mRNA that lie outside the splicing signals play a crucial role in controlling the choice of splicing sites in a given cellular context (reviewed in Refs. 4 and 5).Among these sequences are the splicing enhancers. These elements are found in a wide variety of metazoan pre-mRNAs, either within exons or introns. Purine-rich splicing enhancers (known as ESE) are a well characterized class of exonic splicing enhancers that mostly interact with specific subsets of SR proteins (reviewed in Refs. 6 and 7). SR proteins belong to a family of essential splicing factors that are highly conserved between Drosophila and mammals and that are involved in both constitutive and regulated splicing events (reviewed in Refs. 8 and 9). It has been proposed that the function of SR proteins is to stimulate the recognition of weak upstream 3Ј splice sites, by recruiting U2AF 65/35 , or to f...

The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.