Control of hnRNP A1 alternative splicing: an intron element represses use of the common 3' splice site

Alternative splicing of exon 7B in the hnRNP A1 pre-mRNA produces mRNAs encoding two proteins: hnRNP A1 and the less abundant A1B. We have reported the identification of several intron elements that contribute to exon 7B skipping. In this study, we report the activity of a novel element, conserved element 9 (CE9), located in the intron downstream of exon 7B. We show that multiple copies of CE9 inhibit exon 7B-exon 8 splicing in vitro. When CE9 is inserted between two competing 3' splice sites, a single copy of CE9 decreases splicing to the distal 3' splice site. Our in vivo results also support the conclusion that CE9 is a splicing modulator. First, inserting multiple copies of CE9 into an A1 minigene compromises the production of fully spliced products. Second, one copy of CE9 stimulates the inclusion of a short internal exon in a derivative of the human beta-globin gene. In this case, in vitro splicing assays suggest that CE9 decreases splicing of intron 1, an event that improves splicing of intron 2 and decreases skipping of the short internal exon. The ability of CE9 to act on heterologous substrates, combined with the results of a competition assay, suggest that the activity of CE9 is mediated by a trans-acting factor. Our results indicate that CE9 represses the use of the common 3' splice site in the hnRNP A1 alternative splicing unit.     

Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5' splice site selection in the hnRNP A1 mRNA precursor

In the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 pre-mRNA, different regions in the introns flanking alternative exon 7B have been implicated in the production of the A1 and A1B mRNA splice isoforms. Among these, the CE1a and CE4 elements, located downstream of common exon 7 and alternative exon 7B, respectively, are bound by hnRNP A1 to promote skipping of exon 7B in vivo and distal 5' splice site selection in vitro. Here, we report that CE1a is flanked by an additional high affinity A1 binding site (CE1d). In a manner similar to CE1a, CE1d affects 5' splice site selection in vitro. Consistent with a role for hnRNP A1 in the activity of CE1d, a mutation that abrogates A1 binding abolishes distal 5' splice site activation. Moreover, the ability of CE1d to stimulate distal 5' splice site usage is lost in an HeLa extract depleted of hnRNP A/B proteins, and the addition of recombinant A1 restores the activity of CE1d. Notably, distal 5' splice site selection mediated by A1 binding sites is not compromised in an extract prepared from mouse cells that are severely deficient in hnRNP A1 proteins. In this case, we show that hnRNP A2 compensates for the A1 deficiency. Further studies with the CE4 element reveal that it also consists of two distinct portions (CE4m and CE4p), each one capable of promoting distal 5' splice site use in an hnRNP A1-dependent manner. The presence of multiple A1/A2 binding sites downstream of common exon 7 and alternative exon 7B probably plays an important role in maximizing the activity of hnRNP A1/A2 proteins.     

A novel human nonviral retroposon derived from an endogenous retrovirus.

In a human genome, we found dispersed repetitive sequences homologous to part of a human endogenous retrovirus termed HERV-K which resembled mouse mammary tumor virus. For elucidation of their structure and organization, we cloned some of these sequences from a human gene library. The sequence common to the cloned DNA was ca. 630 base-pairs (bp) in length with an A-rich tail at the 3' end and was found to be a SINE (short interspersed repeated sequence) type nonviral retroposon. In this retroposon, the 5' end had multiple copies of a 40 bp direct repeat very rich in GC content and about the next 510 nucleotides were homologous to the 3' long terminal repeat and its upstream flanking region of the HERV-K genome. This retroposon was thus given the name, SINE-R element since most of it derived from a retrovirus. SINE-R elements were present at 4,000 to 5,000 copies per haploid human genome. The nucleotide sequence was ca. 90% homologous among the cloned elements.