Developmental regulation of neuronal gene expression by Elongator complex protein 1 dosage

Familial dysautonomia(FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in the Elongator complex protein 1(ELP1) gene that leads to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD headed to the discovery that homozygous deletion of the mouse Elp1 gene leads to embryonic lethality prior to mid-gestation. Given that FD is caused by a reduction, not loss, of ELP1, we generated two new mouse models by introducing different c...     

Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator‐dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.     

Urmylation: a ubiquitin-like pathway that functions during invasive growth and budding in yeast

Ubiquitin is a small modifier protein that is conjugated to substrates to target them for degradation. Recently, a surprising number of ubiquitin-like proteins have been identified that also can be attached to proteins. Herein, we identify two molecular functions for the posttranslational protein modifier from Saccharomyces cerevisiae, Urm1p. Simultaneous loss of Urm1p and Cla4p, a p21-activated kinase that functions in budding, is lethal. This result suggests a role for the urmylation pathway in budding. Furthermore, loss of the urmylation pathway causes defects in invasive growth and confers sensitivity to rapamycin. Our results indicate that the sensitivity to rapamycin is due to a genetic interaction with the TOR pathway, which is important for regulation of cell growth in response to nutrients. We have found that Urm1p can be attached to a number of proteins. Loss of five genes that are also essential in a cla4Delta strain, NCS2, NCS6, ELP2, ELP6, and URE2, affect the level of at least one Urm1p conjugate. Moreover, these five genes have a role in invasive growth and display genetic interactions with the TOR pathway. In summary, our results suggest the urmylation pathway is involved in nutrient sensing and budding.     

Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.     

延伸因子复合物对真核生物tRNA的修饰作用

延伸因子复合物(Elongator complex, Elp)由6个亚基蛋白Elp1～6组成,在真核细胞生物中呈现高度的进化保守,提示其具有重要的生物学功能。研究表明,Elp涉及多种细胞行为如转录延伸、细胞外分泌、端粒基因沉默和DNA损伤修复、神经系统的发育和功能等。然而越来越多的证据显示,Elp通过介导tRNA修饰影响翻译过程,从而间接调控上述细胞行为。在人类,ELP1/IKBKAP突变可导致家族性植物神经功能障碍症,ELP2、ELP3和ELP4基因的遗传变异也可能与其他神经退行性病变相关。本文对Elp的结构、Elp修饰tRNA和Elp相关疾病等的研究现状及其进展进行综述。     

Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action

TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1-7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1-6). Unlike TOT1-3 (ELP1-3) and TOT5-7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co-immunoprecipitation, and cell fractionation studies demonstrate partial co-migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P-loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P-loop deletion renders Tot4p biologically inactive.     

Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions

The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm⁵s²U by largely fixing the C3′-endo ribose puckering, ensuring stable and accurate codon–anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm⁵s²U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.     

Identification and expression of the elongator protein 2 (Ajelp2) gene,a novel regeneration-related gene from the sea cucumber Apostichopus japonicus

Elongator proteins comprise six subunits (ELP1–ELP6) and form protein complexes. The elongator protein 2 gene (elp2) encodes a protein with a WD40 repeats domain that acts as a scaffold for complex assembly. It also plays an important role in growth and development. In this study, the full-length cDNA of elongator protein 2 (Ajelp2) was cloned from the sea cucumber Apostichopus japonicus (A. japonicus) using rapid amplification of cDNA ends PCR techniques and comprised 3,058 bp, including a 54 bp 5′ untranslated (UTR), a 526 bp 3′ UTR and a 2,478 bp open reading frame encoding a polypeptide of 825 amino acids. The Ajelp2 sequence showed high homology to 12 other species. The molecular weight and isoelectric of point the presumptive protein were 91.6 kDa and 5.84, respectively. In situ hybridization indicated that the gene is expressed in the body wall, intestine, respiratory tree and longitudinal muscle. The expression level of Ajelp2 increased in recovering of organs in sea cucumber and showed it’s the highest expression level at the 15th day in the intestine and respiratory tree. Its expression then gradually decreased to normal levels. In the body wall, the expression level of Ajelp2 was up-regulated and then down-regulated. These results indicated that Ajelp2 is involved in protein regulation during the regeneration process in the sea cucumber A. japonicus.     

Characterization of a Six-Subunit Holo-Elongator Complex Required for the Regulated Expression of a Group of Genes in Saccharomyces cerevisiae

The Elongator complex associated with elongating RNA polymerase II in Saccharomyces cerevisiae was originally reported to have three subunits, Elp1, Elp2, and Elp3. Using the tandem affinity purification (TAP) procedure, we have purified a six-subunit yeast Holo-Elongator complex containing three additional polypeptides, which we have named Elp4, Elp5, and Elp6. TAP tapping and subsequent purification of any one of the six subunits result in the isolation of all six components. Purification of Elongator in higher salt concentrations served to demonstrate that the complex could be separated into two subcomplexes: one consisted of Elp1, -2, and -3, and the other consisted of Elp4, -5, and -6. Deletions of the individual genes encoding the new Elongator subunits showed that only the ELP5 gene is essential for growth. Disruption of the two nonessential new Elongator-encoding genes, ELP4 and ELP6, caused the same phenotypes observed with knockouts of the original Elongator-encoding genes. Results of microarray analyses demonstrated that the gene expression profiles of strains containing deletions of genes encoding subunits of either Elongator subcomplex, in which we detected significantly altered mRNA expression levels for 96 genes, are very similar, implying that all the Elongator subunits likely function together to regulate a group of S. cerevisiae genes in vivo.     

Analyzing the suppression of respiratory defects in the yeast model of human mitochondrial tRNA diseases

The respiratory defects associated with mutations in human mitochondrial tRNA genes can be mimicked in yeast, which is the only organism easily amenable to mitochondrial transformation. This approach has shown that overexpression of several nuclear genes coding for factors involved in mitochondrial protein synthesis can alleviate the respiratory defects both in yeast and in human cells.     

The Mitochondrial Genome of Raphanus sativus and Gene Evolution of Cruciferous Mitochondrial Types

To explore the mitochondrial genes of the Cruciferae family, the mitochondrial genome of Raphanus sativus (sat) was sequenced and annotated. The circular mitochondrial genome of sat is 239,723 bp and includes 33 protein-coding genes, three rRNA genes and 17 tRNA genes. The mitochondrial genome also contains a pair of large repeat sequences 5.9 kb in length, which may mediate genome reorga-nization into two sub-genomic circles, with predicted sizes of 124.8 kb and 115.0 kb, respectively. Furthermore, gene evolution of mitochondrial genomes within the Cruciferae family was analyzed using sat mitochondrial type (mitotype), together with six other re-ported mitotypes. The cruciferous mitochondrial genomes have maintained almost the same set of functional genes. Compared with Cycas taitungensis (a representative gymnosperm), the mitochondrial genomes of the Cruciferae have lost nine protein-coding genes and seven mitochondrial-like tRNA genes, but acquired six chloroplast-like tRNAs. Among the Cruciferae, to maintain the same set of genes that are necessary for mitochondrial function, the exons of the genes have changed at the lowest rates, as indicated by the numbers of single nucleotide polymorphisms. The open reading frames (ORFs) of unknown function in the cruciferous genomes are not conserved. Evolutionary events, such as mutations, genome reorganizations and sequence insertions or deletions (indels), have resulted in the non- conserved ORFs in the cruciferous mitochondrial genomes, which is becoming significantly different among mitotypes. This work represents the first phylogenic explanation of the evolution of genes of known function in the Cruciferae family. It revealed significant variation in ORFs and the causes of such variation.