Post-transcriptional regulation of gene expression during the programmed death of insect skeletal muscle.

While considerable attention has focused on the role of specific proteins in mediating programmed cell death, few studies have examined the possible involvement of post-transcriptional regulation of mRNAs associated with this developmental process. We have examined developmental changes in transcript stability and translatability using protein extracts generated from the intersegmental muscles (ISM) of the moth Manduca sexta as a cell-free model system to examine three genes that are representative of the patterns of expression observed in condemned ISMs: repressed (actin), induced (polyubiquitin) and constitutively expressed (ubiquitin-fusion 80; ubf80). In addition, we have used luciferase mRNA as a generic reporter transcript to determine if there are sequence-specific controls of mRNA function related to programmed cell death. Among the three Manduca transcripts, polyubiquitin displayed the shortest half-life (t1/2) in all ISM extracts tested. The stability and translatability of all mRNAs were most affected in extracts from muscle cells from day 17 animals, just prior to the commitment of the muscles to die. Transfer of the 3' untranslated regions from the Manduca transcripts to luciferase mRNA did not appreciably change the stability or translatability of this test transcript. These data suggest that there may be global removal of cellular transcripts just prior to death to allow newly expressed mRNAs to rapidly accumulate to high levels. Such changes in message abundance, translatability and stability may facilitate the efficient activation of death (and perhaps other differentiation programs) in some developmental systems.

Lepidopteran DALP, and its mammalian ortholog HIC-5, function as negative regulators of muscle differentiation.

During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes, and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal-transduction molecules that determine whether myoblasts should differentiate or die are largely unknown. In this report, we describe the cloning and characterization of DALP, a small LIM-only type zinc-finger protein that is induced when the intersegmental muscles (ISMs) of the moth Manduca sexta become committed to die at the end of metamorphosis. Forced expression of death-associated LIM-only protein (DALP) in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian ortholog Hic-5, blocks differentiation and induces apoptosis in mouse C(2)C(12) myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die after removal of trophic support. Taken together, these data suggest that DALP and Hic-5 act upstream of MyoD and function as phylogenetically conserved "switches" to block muscle differentiation and induce death.

Sequences and structures required for recombination between virus-associated RNAs.

RNA recombination has been described for a number of viruses in the plant and animal kingdoms, but the mechanisms of selection of recombination sites are poorly understood. The nonrandom recombination between two subviral RNAs associated with turnip crinkle virus was used to study the requirement for specific sequences and structures in the generation of recombinant molecules. Single-base mutations that disrupted either the stem or the loop of one of the two computer-predicted stem-loop structures eliminated detectable recombinant molecules. However, recombinants were detected if compensatory mutations were generated that re-formed a stable hairpin structure. These results provide evidence for the necessity of specific structures in the formation of recombinant molecules in this system.

Recombination between satellite and genomic RNAs of turnip crinkle virus.

New recombinant molecules formed from satellite and genomic RNAs of turnip crinkle virus (TCV) have been characterized. Known collectively as sat-RNA CX, these molecules are composed of a nearly full-length segment of a previously characterized TCV satellite RNA (sat-RNA D) at the 5' end joined to variable lengths of TCV genomic RNA 3' terminal sequence. Sat-RNA CX molecules fall into two classes: molecules of 420 to 435 bases and larger species of 501 to 506 bases. The TCV sequence at the junction of the larger molecules is purine-rich and is similar to a motif found at the 5' ends of the TCV satellite RNAs and at the junctions of some TCV defective interfering RNAs. The TCV sequence at the junction of the smaller sat-RNA CX molecules is pyrimidine-rich and is similar to the sequence at the right side of a junction of one TCV defective interfering RNA as well as sequence immediately downstream of the internal initiation site of the 1.45-kb TCV subgenomic RNA. We propose that the latter motif is another putative signal recognized by the viral replicase during the generation of defective interfering and recombinant RNAs in the TCV system.

Formation of multimers of linear satellite RNAs.

A 22-base region of turnip crinkle virus satellite-RNA C (sat-RNA C) is involved in the accumulation of monomeric and dimeric forms. Deletions within the region inhibited the accumulation of sat-RNA C monomers. However, normal ratios of dimers to monomers occurred if the 22 bases were replaced by 22 unrelated bases or if the location of this region was altered. Therefore, these specific 22 bases are not involved in the accumulation of sat-RNA C monomers. Examination of the sequences at the junctions of multimers of all three turnip crinkle virus sat-RNAs revealed the deletion of bases corresponding to the 3' and 5' ends of monomeric units as well as the addition of nucleotides not present in monomers. Based on these results, we present a model to explain the formation of multimers of linear subviral RNAs associated with turnip crinkle virus. Our model suggests that multimers are formed by the reinitiation of replication by the replicase before release of the nascent strand. We have previously proposed the same mechanism for the formation of defective interfering RNAs, chimeric sat-RNAs, and sat-RNA recombinants in the turnip crinkle virus system (Cascone, Carpenter, Li, and Simon. (1990). EMBO J. 9, 1709-1715).

Mutations in a satellite RNA of turnip crinkle virus result in addition of poly(U) in vivo.

Turnip crinkle virus (TCV) is associated with many subviral RNAs including satellite (sat-) RNAs which require a helper virus for infectivity. When plants were inoculated with TCV and transcripts of TCV sat-RNA C containing deletions of 3 to 8 nucleotides beginning at position 100 and extending toward the 5' end, some of the sat-RNA isolated from plants migrated more slowly than expected on denaturing polyacrylamide gels. Cleavage of the sat-RNA into two segments by digestion with RNase H following hybridization to an oligonucleotide complementary to internal sat-RNA sequence indicated that the 5' one-third of the molecule was involved in the abnormal gel migration. Sat-RNAs derived from transcripts with a deletion of bases in position 96-100 were cloned. Sequencing of the cDNAs revealed that the aberrant migration of the sat-RNAs was due to the presence of variable lengths of poly(U) 10 nucleotides downstream from the deletion at a position which already contained five U residues. Deletions extending toward the 3' end in the same region did not result in poly(U) additions. Mutations in the original five U residues along with the 5' deletions also did not lead to poly(U) additions. The insertion of poly(U) in TCV sat-RNA C may be a new example of replicase stuttering with the distinction that it only occurs following specific upstream mutations.

Recombination between satellite RNAs of turnip crinkle virus.

Turnip crinkle virus (TCV) is associated with satellite (sat) RNAs (sat-RNA D, sat-RNA F), defective interfering (DI) RNAs (DI RNA G, DI1 RNA), and one RNA with properties of both sat-RNAs and DI RNAs (sat-RNA C). When plants were inoculated with TCV, sat-RNA D and in vitro sat-RNA C transcripts containing non-viable mutations in the 5' domain, recombinant sat-RNAs were recovered. These recombinants were composed of sat-RNA D at the 5' end and sat-RNA C sequences at the 3' end. Analysis of 20 independent recombination junctions revealed that unequal crossing-over had occurred in planta in a region of sequence similarity between the two sat-RNAs which resulted in the duplication of 3-16 nucleotides. Thirty percent of the sat-RNA recombinants also had one to three additional nucleotides inserted at the crossover junctions which did not correspond to either sat-RNA C or sat-RNA D sequence. The right side of the recombination junctions always began with one of three consecutive nucleotides of sat-RNA C. Based on the similarity between this sequence of sat-RNA C, the right side junction of DI RNA G and the 5' end of TCV, as well as the sequence similarity between right side junctions of DI1 RNA and sat-RNA C and the 5' end of the sat-RNAs, a replicase-driven copy choice mechanism is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)