Zanamivir: an alternative translation.

This paper offers an alternative view to that given elsewhere regarding the value of zanamivir as an agent for treating patients who develop influenza symptoms. The position taken here has developed as a result of an analysis of the data that was undertaken by the journal Drug and Therapeutics Bulletin.     

Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short‐read RNA sequencing, single molecule long‐read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron‐containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non‐conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.     

Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies.

Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.     

Noncircadian regulation and function of clock genes period and timeless in oogenesis of Drosophila melanogaster

Circadian clock genes are ubiquitously expressed in the nervous system and peripheral tissues of complex animals. While clock genes in the brain are essential for behavioral rhythms, the physiological roles of these genes in the periphery are not well understood. Constitutive expression of the clock gene period was reported in the ovaries of Drosophila melanogaster; however, its molecular interactions and functional significance remained unknown. This study demonstrates that period (per) and timeless (tim) are involved in a novel noncircadian function in the ovary. PER and TIM are constantly expressed in the follicle cells enveloping young oocytes. Genetic evidence suggests that PER and TIM interact in these cells, yet they do not translocate to the nucleus. The levels of TIM and PER in the ovary are affected neither by light nor by the lack of clock-positive elements Clock (Clk) and cycle (cyc). Taken together, these data suggest that per and tim are regulated differently in follicle cells than in clock cells. Experimental evidence suggests that a novel fitness-related phenotype may be linked to noncircadian expression of clock genes in the ovaries. Mated females lacking either per or tim show nearly a 50% decline in progeny, and virgin females show a similar decline in the production of mature oocytes. Disruption of circadian mechanism by either the depletion of TIM via constant light treatment or continuous expression of PER via GAL4/UAS expression system has no adverse effect on the production of mature oocytes.     

The translation factors of Drosophila melanogaster

Synthesis of polypeptides from mRNA (translation) is a fundamental cellular process that is coordinated and catalyzed by a set of canonical ‘translation factors’. Surprisingly, the translation factors of Drosophila melanogaster have not yet been systematically identified, leading to inconsistencies in their nomenclature and shortcomings in functional (Gene Ontology, GO) annotations. Here, we describe the complete set of translation factors in D. melanogaster, applying nomenclature already in widespread use in other species, and revising their functional annotation. The collection comprises 43 initiation factors, 12 elongation factors, 3 release factors and 6 recycling factors, totaling 64 of which 55 are cytoplasmic and 9 are mitochondrial. We also provide an overview of notable findings and particular insights derived from Drosophila about these factors. This catalog, together with the incorporation of the improved nomenclature and GO annotation into FlyBase, will greatly facilitate access to information about the functional roles of these important proteins.     

Isolation and analysis of six timeless alleles that cause short- or long-period circadian rhythms in Drosophila

In genetic screens for Drosophila mutations affecting circadian locomotion rhythms, we have isolated six new alleles of the timeless (tim) gene. Two of these mutations cause short-period rhythms of 21-22 hr in constant darkness, and four result in long-period cycles of 26-28 hr. All alleles are semidominant. Studies of the genetic interactions of some of the tim alleles with period-altering period (per) mutations indicate that these interactions are close to multiplicative; a given allele changes the period length of the genetic background by a fixed percentage, rather than by a fixed number of hours. The tim(L1) allele was studied in molecular detail. The long behavioral period of tim(L1) is reflected in a lengthened molecular oscillation of per and tim RNA and protein levels. The lengthened period is partly caused by delayed nuclear translocation of TIM(L1) protein, shown directly by immunocytochemistry and indirectly by an analysis of the phase response curve of tim(L1) flies.     

Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock.

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the PERIOD protein. Whereas the period (per), timeless (tim), and dClock (dClk) genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and DBT protein are constitutively expressed. Robust circadian changes in DBT subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include PER, TIM, and DBT, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of PER is strongly enhanced when TIM is removed from PER/TIM/DBT complexes. The varying associations of PER, DBT and TIM appear to determine the onset and duration of nuclear PER function within the Drosophila clock.     

A consideration of alternative models for the initiation of translation in eukaryotes.

Although recent biochemical and genetic investigations have produced some insights into the mechanism of initiation of translation in eukaryotic cells, two aspects of the initiation process remain controversial. One unsettled issue concerns a variety of functions that have been proposed for mRNA binding proteins, including some initiation factors. The need to distinguish between specific and nonspecific binding of proteins to mRNA is discussed herein. The possibility that certain initiation factors might act as RNA helicases is evaluated along with other ideas about the functions of mRNA- and ATP-binding factors. A second controversial issue concerns the universality of the scanning mechanism for initiation of translation. According to the conventional scanning model, the initial contact between eukaryotic ribosomes and mRNA occurs exclusively at the 5' terminus of the message, which is usually capped. The existence of uncapped mRNAs among a few plant and animal viruses has prompted a vigorous search for other modes of initiation. An "internal initiation" mechanism, first proposed for picornaviruses, has received considerable attention. Although a large body of evidence has been adduced in support of such a mechanism, many of the experiments appear flawed or inconclusive. Some suggestions are given for improving experiments designed to test the internal initiation hypothesis.