Zfrp8 forms a complex with fragile-X mental retardation protein and regulates its localization and function.

Fragile-X syndrome is the most commonly inherited cause of autism and mental disabilities. The Fmr1 (Fragile-X Mental Retardation 1) gene is essential in humans and Drosophila for the maintenance of neural stem cells, and Fmr1 loss results in neurological and reproductive developmental defects in humans and flies. FMRP (Fragile-X Mental Retardation Protein) is a nucleo-cytoplasmic shuttling protein, involved in mRNA silencing and translational repression. Both Zfrp8 and Fmr1 have essential functions in the Drosophila ovary. In this study, we identified FMRP, Nufip (Nuclear Fragile-X Mental Retardation Protein-interacting Protein) and Tral (Trailer Hitch) as components of a Zfrp8 protein complex. We show that Zfrp8 is required in the nucleus, and controls localization of FMRP in the cytoplasm. In addition, we demonstrate that Zfrp8 genetically interacts with Fmr1 and tral in an antagonistic manner. Zfrp8 and FMRP both control heterochromatin packaging, also in opposite ways. We propose that Zfrp8 functions as a chaperone, controlling protein complexes involved in RNA processing in the nucleus.

Zfrp8/PDCD2 Interacts with RpS2 Connecting Ribosome Maturation and Gene-Specific Translation.

Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Our previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (TEs). Here we show that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and TE transcripts show increased nuclear accumulation. We also show that Zfrp8/PDCD2 interacts with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins we demonstrate that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. Our results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation.

Discs large 5, an Essential Gene in Drosophila, Regulates Egg Chamber Organization.

Discs large 5 (Dlg5) is a member of the MAGUK family of proteins that typically serve as molecular scaffolds and mediate signaling complex formation and localization. In vertebrates, Dlg5 has been shown to be responsible for polarization of neural progenitors and to associate with Rab11-positive vesicles in epithelial cells. In Drosophila, however, the function of Dlg5 is not well-documented. We have identified dlg5 as an essential gene that shows embryonic lethality. dlg5 embryos display partial loss of primordial germ cells (PGCs) during gonad coalescence between stages 12 and 15 of embryogenesis. Loss of Dlg5 in germline and somatic stem cells in the ovary results in the depletion of both cell lineages. Reduced expression of Dlg5 in the follicle cells of the ovary leads to a number of distinct phenotypes, including defects in egg chamber budding, stalk cell overgrowth, and ectopic polar cell induction. Interestingly, loss of Dlg5 in follicle cells results in abnormal distribution of a critical component of cell adhesion, E-cadherin, shown to be essential for proper organization of egg chambers.

Temporal regulation of viral transcription during development of Thermus thermophilus bacteriophage phiYS40.

Regulation of gene expression of lytic bacteriophage varphiYS40 that infects the thermophilic bacterium Thermus thermophilus was investigated and three temporal classes of phage genes, early, middle, and late, were revealed. varphiYS40 does not encode a (RNAP) and must rely on host RNAP for transcription of its genes. Bioinformatic analysis using a model of Thermus promoters predicted 43 putative sigma(A)-dependent -10/-35 class phage promoters. A randomly chosen subset of those promoters was shown to be functional in vivo and in vitro and to belong to the early temporal class. Macroarray analysis, primer extension, and bioinformatic predictions identified 36 viral middle and late promoters. These promoters have a single common consensus element, which resembles host sigma(A) RNAP holoenzyme -10 promoter consensus element sequence. The mechanism responsible for the temporal control of the three classes of promoters remains unknown, since host sigma(A) RNAP holoenzyme purified from either infected or uninfected cells efficiently transcribed all varphiYS40 promoters in vitro. Interestingly, our data showed that during infection, there is a significant increase and decrease of transcript amounts of host translation initiation factors IF2 and IF3, respectively. This finding, together with the fact that most middle and late varphiYS40 transcripts were found to be leaderless, suggests that the shift to late viral gene expression may also occur at the level of mRNA translation.

Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions.

We determined the sequence of the 152,372 bp genome of phiYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of phiYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. phiYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of phiYS40, most of which have no similarity to sequences in public databases, were identified by mass spectrometric analysis of purified virions. Various phiYS40 proteins have different phylogenetic neighbors, including myovirus, podovirus, and siphovirus gene products, bacterial genes and, in one case, a dUTPase from a eukaryotic virus. phiYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.

The role of the largest RNA polymerase subunit lid element in preventing the formation of extended RNA-DNA hybrid.

Analysis of multi-subunit RNA polymerase (RNAP) structures revealed several distinct elements that may perform partial functions of the enzyme. One such element, the "lid", is formed by an evolutionarily conserved segment of the RNAP largest subunit (beta' in bacterial RNAP). The beta' lid contacts the nascent RNA at the upstream edge of the RNA-DNA hybrid, where the RNA gets separated from the DNA template-strand and double-stranded upstream DNA is formed. To test the beta' lid functions, we generated bacterial RNAP lacking the lid and studied the mutant enzyme's properties in vitro. Our results demonstrate that removal of the lid has minimal consequences on transcription elongation from double-stranded DNA. On single-stranded DNA, the mutant RNAP generates full-sized transcripts that remain annealed to the DNA throughout their length. In contrast, the wild-type enzyme produces short, 18-22 nucleotide transcripts that remain part of the transcription complex but cannot be further elongated. The cessation of transcription is apparently triggered by a clash between the lid and the nascent RNA 5' end. The results show that the lid's function is redundant in the presence of the non-template DNA strand, which alone can control the proper geometry of nucleic acids at the upstream edge of the transcription complex. Structural considerations suggest that in the absence of the non-template strand and the lid, a new channel opens within the RNAP molecule that allows continuous DNA-RNA hybrid to exit RNAP.

The mechanism of DNA replication primer synthesis by RNA polymerase.

RNA primers for DNA replication are usually synthesized by specialized enzymes, the primases. However, some replication systems have evolved to use cellular DNA-dependent RNA polymerase for primer synthesis. The main requirement for the replication primer, an exposed RNA 3' end annealed to the DNA template, is not compatible with known conformations of the transcription elongation complex, raising a question of how the priming is achieved. Here we show that a previously unrecognized kind of transcription complex is formed during RNA polymerase-catalysed synthesis of the M13 bacteriophage replication primer. The complex contains an overextended RNA-DNA hybrid bound in the RNA-polymerase trough that is normally occupied by downstream double-stranded DNA, thus leaving the 3' end of the RNA available for interaction with DNA polymerase. Transcription complexes with similar topology may prime the replication of other bacterial mobile elements and may regulate transcription elongation under conditions that favour the formation of an extended RNA-DNA hybrid.

Role of second-largest RNA polymerase I subunit Zn-binding domain in enzyme assembly.

The second-largest subunits of eukaryal RNA polymerases are similar to the beta subunits of prokaryal RNA polymerases throughout much of their lengths. The second-largest subunits from eukaryal RNA polymerases contain a four-cysteine Zn-binding domain at their C termini. The domain is also present in archaeal homologs but is absent from prokaryal homologs. Here, we investigated the role of the C-terminal Zn-binding domain of Rpa135, the second-largest subunit of yeast RNA polymerase I. Analysis of nonfunctional Rpa135 mutants indicated that the Zn-binding domain is required for recruitment of the largest subunit, Rpa190, into the RNA polymerase I complex. Curiously, the essential function of the Rpa135 Zn-binding domain is not related to Zn(2+) binding per se, since replacement of only one of the four cysteine residues with alanine led to the loss of Rpa135 function. Even more strikingly, replacement of all four cysteines with alanines resulted in functional Rpa135.