Control of alternative pre-mRNA splicing by distributed pentameric repeats

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.

Vaccinia virus DNA replication: two hundred base pairs of telomeric sequence confer optimal replication efficiency on minichromosome templates.

Vaccinia virus is a complex DNA virus that exhibits significant genetic and physical autonomy from the host cell. Most if not all of the functions involved in replication and transcription of the 192-kb genome are virally encoded. Although significant progress has been made in identifying trans-acting factors involved in DNA synthesis, the mechanism of genome replication has remained poorly understood. The genome is a linear duplex with covalently closed hairpin termini, and it has been presumed that sequences and/or structures within these termini are important for the initiation of genome replication. In this report we describe the construction of minichromosomes containing a central plasmid insert flanked by hairpin termini derived from the viral genome and their use as replication templates. When replication of these minichromosomes was compared with a control substrate containing synthetic hairpin termini, specificity for viral telomeres was apparent. Inclusion of > or = 200 bp from the viral telomere was sufficient to confer optimal replication efficiency, whereas 65-bp telomeres were not effective. Chimeric 200-bp telomeres containing the 65-bp terminal element and 135 bp of ectopic sequence also failed to confer efficient replication, providing additional evidence that telomere function is sequence-specific. Replication of these exogenous templates was dependent upon the viral replication machinery, was temporally coincident with viral replication, and generated covalently closed minichromosome products. These data provide compelling evidence for specificity in template recognition and utilization in vaccinia virus-infected cells.