TWO ALTERNATIVELY SPLICED TRANSCRIPTS FROM THE DROSOPHILA PERIOD GENE RESCUE RHYTHMS HAVING DIFFERENT MOLECULAR AND BEHAVIORAL CHARACTERISTICS

The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The per gene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the three per mRNA types were based on the analysis of cDNA clones, we tested whether these mRNA types were present in vivo by RNase protection assays and reverse transcriptasemediated PCR. The results show that per generates two transcript types that differ only by the presence (type A) or absence (type B*) of an alternative intron in the 3* untranslated region. Transgenic flies containing transgenes that produce only type B* transcripts (per B* ), type A transcripts (per A ), or both transcripts (per G ) rescue locomotor activity rhythms with average periods of 24.7, 25.4, and 24.4 h, respectively. Although no appreciable differences in type A and type B* mRNA cycling were observed, a slower accumulation of PER in flies making only type A transcripts suggests that the intron affects the translation of per mRNA. The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The expression of these genes is required for circadian clock function, and an important aspect of their expression is circadian fluctuations in their mRNA and protein levels (17). These rhythms in per and tim gene products are controlled by a circadian feedback loop in which PER and TIM proteins control the expression of their own mRNAs (17, 25, 26). This feedback is mediated predominantly at the transcriptional level, though posttranscriptional regulation is also involved (5, 16, 29, 31, 32). The role of PER in this process is unknown, but its lack of a known DNA binding domain and inability to bind DNA indicate that it does not regulate transcription directly (17, 25). Analysis of per cDNA clones uncovered three splice variants that encode three different PER isoforms (3). The most abundant of these transcripts, type A, defined both the structure of the per gene and the prototypical 1,218-amino-acid PER protein. Type B transcripts differ from type A by having two additional introns; one removes 288 nucleotides (nt) from exon 5 of type A transcripts, and the other excises 89 nt from the 39 untranslated region (39UTR) of exon 8. After excising the intron from exon 5, type B transcripts produce a protein that is 96 amino acids shorter. The least abundant transcript (only one partial cDNA clone was isolated), type C, differs from type A by retaining introns 5, 6, and 7, thereby producing a transcript whose exon 5 spans exons 5 to 8 in type A transcripts. Due to the inclusion of these additional introns, the last 107 amino acids of the putative type C protein sequence are entirely different from the last 149 amino acids of type A protein sequence. All three per cDNAs are capable of rescuing behavioral rhythms in per 01 flies, though the type C construct may mediate behavioral rescue by generating both type A and type B transcripts (3, 4). Given the critical role that PER plays in controlling the circadian feedback loop in Drosophila, it is important to determine which isoforms contribute to the feedback loop mechanism and what impact this contribution may have on behavioral rhythms. Since the initial characterization of per mRNA splice variants was based on the structure and abundance of partial cDNA clones, we tested whether these per transcripts exist in vivo and function equally to rescue locomotor activity rhythms. Our studies failed to detect per splice variants that generate different PER isoforms. However, two per transcripts that differ by an alternatively spliced intron within their 39UTRs were found; type A contains the 89-bp intron, and type B9 lacks this intron. Transgenes that produce type A mRNA, type B9 mRNA, or both mRNA types each rescue robust locomotor activity rhythms, but the period of these rhythms tends to be longer in the transgene that produces only type A transcripts. Type A and type B9 transcripts are indistinguishable with respect to circadian cycling, but the levels of PER derived from the transgene expressing only type A transcripts rise with a later phase than PER derived from transgenes expressing only type B9 or both type A and type B9 transcripts. These results suggest that the alternatively spliced intron alters the translation of per mRNA.