Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells.

By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukaryotic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocalize in yeast and human cells.

Modulation of gene expression via overlapping binding sites exerted by ZNF143, Notch1 and THAP11.

ZNF143 is a zinc-finger protein involved in the transcriptional regulation of both coding and non-coding genes from polymerase II and III promoters. Our study deciphers the genome-wide regulatory role of ZNF143 in relation with the two previously unrelated transcription factors Notch1/ICN1 and thanatos-associated protein 11 (THAP11) in several human and murine cells. We show that two distinct motifs, SBS1 and SBS2, are associated to ZNF143-binding events in promoters of >3000 genes. Without co-occupation, these sites are also bound by Notch1/ICN1 in T-lymphoblastic leukaemia cells as well as by THAP11, a factor involved in self-renewal of embryonic stem cells. We present evidence that ICN1 binding overlaps with ZNF143 binding events at the SBS1 and SBS2 motifs, whereas the overlap occurs only at SBS2 for THAP11. We demonstrate that the three factors modulate expression of common target genes through the mutually exclusive occupation of overlapping binding sites. The model we propose predicts that the binding competition between the three factors controls biological processes such as rapid cell growth of both neoplastic and stem cells. Overall, our study establishes a novel relationship between ZNF143, THAP11 and ICN1 and reveals important insights into ZNF143-mediated gene regulation.

Genome-wide evidence for an essential role of the human Staf/ZNF143 transcription factor in bidirectional transcription.

In the human genome, ∼ 10% of the genes are arranged head to head so that their transcription start sites reside within <1 kbp on opposite strands. In this configuration, a bidirectional promoter generally drives expression of the two genes. How bidirectional expression is performed from these particular promoters constitutes a puzzling question. Here, by a combination of in silico and biochemical approaches, we demonstrate that hStaf/ZNF143 is involved in controlling expression from a subset of divergent gene pairs. The binding sites for hStaf/ZNF143 (SBS) are overrepresented in bidirectional versus unidirectional promoters. Chromatin immunoprecipitation assays with a significant set of bidirectional promoters containing putative SBS revealed that 93% of them are associated with hStaf/ZNF143. Expression of dual reporter genes directed by bidirectional promoters are dependent on the SBS integrity and requires hStaf/ZNF143. Furthermore, in some cases, functional SBS are located in bidirectional promoters of gene pairs encoding a noncoding RNA and a protein gene. Remarkably, hStaf/ZNF143 per se exhibits an inherently bidirectional transcription activity, and together our data provide the demonstration that hStaf/ZNF143 is indeed a transcription factor controlling the expression of divergent protein-protein and protein-non-coding RNA gene pairs.

The scaRNA2 is produced by an independent transcription unit and its processing is directed by the encoding region.

The C/D box scaRNA2 is predicted to guide specific 2'-O-methylation of U2 snRNA. In contrast to other SCARNA genes, SCARNA2 appears to be independently transcribed. By transient expression of SCARNA2-reporter gene constructs, we have demonstrated that this gene is transcribed by RNA polymerase II and that the promoter elements responsible for its transcription are contained within a 161 bp region upstream of the transcription start site. In mammals, we have identified four cross species conserved promoter elements, a TATA motif, an hStaf/ZNF143 binding site and two novel elements that are required for full promoter activity. Binding of the human hStaf/ZNF143 transcription factor to its target sequence is required for promoter activity, suggesting that hStaf/ZNF143 is a fundamental regulator of the SCARNA2 gene. We also showed that RNA polymerase II continues transcription past the 3'-end of the mature RNA, irrespective of the identity of the Pol II promoter. The 3'-end processing and accumulation are governed by the sole information contained in the scaRNA2 encoding region, the maturation occurring via a particular pathway incompatible with that of mRNA or snRNA production.

Transcription of the human cell cycle regulated BUB1B gene requires hStaf/ZNF143.

BubR1 is a key protein mediating spindle checkpoint activation. Loss of this checkpoint control results in chromosomal instability and aneuploidy. The transcriptional regulation of the cell cycle regulated human BUB1B gene, which encodes BubR1, was investigated in this report. A minimal BUB1B gene promoter containing 464 bp upstream from the translation initiation codon was sufficient for cell cycle regulated promoter activity. A pivotal role for transcription factor hStaf/ZNF143 in the expression of the BUB1B gene was demonstrated through gel retardation assays, transient expression of mutant BUB1B promoter-reporter gene constructs and chromatin immunoprecipitation assay. Two phylogenetically conserved hStaf/ZNF143-binding sites (SBS) were identified which are indispensable for BUB1B promoter activity. In addition, we found that the domain covering the transcription start sites contains conserved boxes homologous to initiator (Inr), cell cycle dependent (CDE) and cell cycle genes homology regions (CHR) elements. Mutations within the CDE and CHR elements led to diminished cell cycle regulation of BUB1B transcription. These results demonstrate that BUB1B gene transcription is positively regulated by hStaf/ZNF143, a ubiquitously expressed factor, and that the CDE-CHR tandem element was essential for G2/M-specific transcription of the BUB1B gene.

Characterization of snRNA and snRNA-type genes in the pufferfish Fugu rubripes.

Vertebrate snRNA and snRNA-type genes occur in independent transcription units with external promoters. The transcription level from the basal promoter is enhanced by the distal sequence element DSE. This element contains almost invariably two activator submotifs, the Staf binding site and the octamer motif, recruiting the Staf and Oct-1 transcriptional activators. In the present work, database search identified 35 snRNA and snRNA-type genes in the genome sequence of the pufferfish Fugu rubripes. Sequence comparisons of promoter elements, determination of template activities by microinjection into Xenopus oocytes and DNA binding assays of the transcriptional activators led to the surprising finding that only two Fugu genes conform to the general scheme with the expected two submotifs in the DSE. Distinctively, all the other DSEs harbor a unique Staf binding site. Also striking was the observation that the tRNA(Sec), and the snRNA genes that are tandemly repeated, are transcribed from promoter-less DSEs. Evolutionary implications of these results are discussed.

A genome scale location analysis of human Staf/ZNF143-binding sites suggests a widespread role for human Staf/ZNF143 in mammalian promoters.

Staf was originally identified as the transcriptional activator of Xenopus tRNA(Sec) and small nuclear (sn) RNA-type genes. Recently, transcription of seven human (h) protein coding genes was reported to be activated by the human ortholog hStaf/ZNF143. Here we have used a combined in silico and biochemical approach to identify 1175 conserved hStaf/ZNF143-binding sites (SBS) distributed in 938 promoters of four mammalian genomes. The SBS shows a significant positional preference and occurs mostly within 200 bp upstream of the transcription start site. Chromatin immunoprecipitation assays with 295 of the promoters established that 90% contain bona fide SBS. By extrapolating the values of this mapping to the full sizes of the mammalian genomes, we can infer the existence of at least 2500 SBS distributed in 2000 promoters. This unexpected large number strongly suggests that SBS constitutes one of the most widespread transcription factor-binding sites in mammalian promoters. Furthermore, we demonstrated that the presence of the SBS alone is sufficient to direct expression of a luciferase reporter gene, suggesting that hStaf/ZNF143 can recruit per se the transcription machinery.