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Prediction and verification of mouse tRNAs identifies intron-containing families

ABSTRACT: Computer algorithms are often used to identify tRNA genes in newly sequenced genomes, but these predictions can be challenging. Not only are there structural variations and extremely limited sequence conservation among genes, but vertebrate genomes tend to have highly reiterated short interspersed sequences (SINEs) that originally derived from tRNA genes or tRNA-like transcription units. We have employed two programs, tRNAScan SE and ARAGORN, to predict the tRNA genes in the mouse nuclear genome, resulting in quite diverse but overlapping predicted gene sets. From these, we removed known SINE repeats and sorted the genes into predicted families and single-copy genes. In particular, four families of intron-containing tRNA genes were predicted, with introns in positions and structures analogous to the well characterized intron-containing tRNA genes in yeast. In this work we focus on verifying the expression of the intron-containing tRNA gene familes, as well as the other 30 tRNA gene familes. Keywords: tRNA, direct label

ORGANISM(S): Mus musculus

PROVIDER: GSE8224 | GEO | 2008/02/22

SECONDARY ACCESSION(S): PRJNA101149

REPOSITORIES: GEO

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Mus musculus

Project description:Prediction and verification of mouse tRNAs identifies intron-containing families

| PRJNA101149 | ENA

Protein ID of ϕXacN1

Project description:ϕXacN1 is a novel jumbo myovirus infecting the causative agent of Asian citrus canker, Xanthomonas citri. Its linear 384,670 bp double-stranded DNA genome encodes 592 predicted protein coding genes and shows 65,875 bp direct terminal repeats (DTRs), so far the longest DTRs among sequence phage genomes. The DTRs harbor 56 tRNA genes, corresponding to all 20 amino acids. This is the highest number of tRNA genes reported in a phage genome. Codon usage analyses revealed a propensity that the phage encoded tRNAs target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes, additional seven translation-related genes as well as a chaperonin gene found in the ϕXacN1 genome suggests an increased level of independence of phage replication on host molecular machinery and a wide host range. Consistently, ϕXacN1 showed a wider host range than other X. citri phages in an infection test against a panel of X. citri strains. Phylogenetic analyses revealed a clade of phages composed of ϕXacN1 and ten other jumbo phages showing an evolutionary stability in their large genome sizes.

2018-10-30 | PXD008065 | JPOST Repository

Characterizing expression and processing of precursor and mature human tRNAs by hydro-tRNAseq and PAR-CLIP

Project description:The participation of transfer RNAs (tRNAs) in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes, and curation of precursor and mature tRNA sequences. This has been challenging, mainly because RNA secondary structure and nucleotide modifications, together with tRNA gene multiplicity, complicate sequencing and sequencing read mapping efforts. To address these issues, we developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing. To identify transcribed tRNA genes, we further complemented this approach with Photoactivatable Crosslinking and Immunoprecipitation (PAR-CLIP) of SSB/La, a conserved protein involved in pre-tRNA processing. Our results show that approximately half of all predicted tRNA genes are transcribed in human cells. We also report predominant nucleotide modification sites, their order of introduction, and identify tRNA leader, trailer and intron sequences. By using complementary sequencing-based methodologies, we present a human tRNA atlas, and determine expression levels of mature and processing intermediates of tRNAs in human cells.

2017-07-17 | GSE95683 | GEO

The Clp1 R140H mutation alters tRNA metabolism and mRNA 3’ processing in mouse models of pontocerebellar hypoplasia

Project description:Homozygous mutation of the RNA kinase CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the tRNA splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments upregulated, and other fragments downregulated. However, the spatiotemporal patterns of dysregulation did not support a role in pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on pre-mRNA cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes also were expressed at lower levels in the Clp1R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that the contribution of mRNA 3’ processing to pathogenesis in PCH10 merits further investigation.

2021-09-20 | GSE176183 | GEO

Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing

Project description:Background: The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA, remain elusive. Results: Employing in silico analysis we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach we show that a partial deletion of a single endogenous nimtRNALys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3’-splice site associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNATyr by an electrophoretic mobility shift assay. Conclusions: We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.

2020-10-28 | PXD022204 | Pride

Differential Recruitment of the Splicing Machinery during Transcription Predicts Genome-wide Patterns of mRNA Splicing

Project description:The splicing machinery associates with genes to facilitate efficient co-transcriptional mRNA processing. We have mapped these associations by genome localization analysis to ascertain how splicing is achieved and regulated on a system-wide scale. Our data show that factors important for intron recognition sample nascent mRNAs and are retained specifically at intron-containing genes via RNA-dependent interactions. Spliceosome assembly proceeds co-transcriptionally, but completes post-transcriptionally in most cases. Some intron-containing genes were not bound by the spliceosome, including several developmentally regulated genes. On this basis we predicted and verified regulated splicing, and observed a role for nuclear mRNA surveillance in monitoring those events. Finally, we present evidence that co-transcriptional processing events determine the recruitment of specific mRNA export factors. Broadly, our results provide mechanistic insights into the coordinated regulation of transcription, mRNA processing, and nuclear export in executing complex gene expression programs. Keywords: ChIP-chip

2006-12-13 | GSE6480 | GEO

RTP801 INTERACTS WITH THE tRNA LIGASE COMPLEX AND DYSREGULATES ITS RNA LIGASE ACTIVITY IN ALZHEIMER’S DISEASE

Project description:RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer’s disease (AD) that contributes to cognitive deficits and neuroinflammation. Here we found that RTP801 interacts with HSPC117, DDX1, and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro, since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. On the contrary, RTP801 overexpression inhibited the splicing of XBP1. In this line, in human AD postmortem hippocampal samples, where RTP801 protein levels are upregulated, we found that XBP1 splicing dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo, in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach.

2024-09-12 | GSE267524 | GEO

Microarray analysis of Xenopus tRNA isoacceptor families, RNA Polymerase III target genes and selected mRNAs in control and fgf4/wnt8 induced animal cap explants at NF stage 25

Project description:Animal poles of 1 to 2-cell stage embryos were injected with a cocktail containing 5pg of Fgf4 and 50pg wnt8 mRNA. Embryos were grown alongside batch controls to NF stage 8/9. Animal cap explants were dissected from the embryos and grown overnight in NAM alongside whole embryo controls as a means of staging. At NF stage 25, caps were collected for microarray analysis. Custom microarrays containing sequences for Xenopus tRNA and RNA Polymerase III gene families as well as selected mRNAs were used to detect changes in transcription at this stage in response to induction of myogenic differentiation. We aimed to use this data to investigate the relationship between any codon bias in highly expressed mRNAs with expression of their matching tRNA isoacceptors in response to a specific differentiation programme.

2019-09-30 | E-MTAB-7752 | biostudies-arrayexpress

Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA splicing in pontocerebellar hypoplasia

Project description:Our work defines the molecular principles of intron excision in humans and provides evidence that destabilized TSEN contributes to a PCH phenotype by impairing tRNA splicing at the intron-excision step.

2021-06-18 | GSE151236 | GEO

RNA-seq mouse development

Project description:The genetic code is an abstraction of how mRNA codons and tRNA anticodons molecularly interact during protein synthesis; the stability and regulation of this interaction remains largely unexplored. Here, we quantitatively characterized the expression of mRNA and tRNA genes across developing mouse tissues. Substantial fractions of both gene sets dynamically change expression from early organogenesis to adult tissues. mRNA codon pools are highly stable over development and reflect the genomic background; in contrast, changes in transcription at specific tRNA genes are coordinated across anticodon families to produce a stable isoacceptor output. During development, the pools of mRNA codons and tRNA anticodons are invariant and highly correlated, revealing a stable molecular interaction interlocking transcription and translation.

2014-08-08 | E-MTAB-2328 | biostudies-arrayexpress

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