scholarly journals Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Simon M. Hoser ◽  
Anne Hoffmann ◽  
Andreas Meindl ◽  
Maximilian Gamper ◽  
Jörg Fallmann ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Human Genome ◽  
Splice Site ◽  
Mutational Analysis ◽  
Mitochondrial Protein ◽  
Regulatory Elements ◽  
Trna Genes ◽  
Mitochondrial Trna ◽  
Mobility Shift ◽  
Cloverleaf Structure

Abstract 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 the 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.

scholarly journals Mitochondrial tRNA mutations in Chinese children with tic disorders

2020 ◽  
Vol 40 (12) ◽  
Author(s):  
Peifang Jiang ◽  
Yinjie Ling ◽  
Tao Zhu ◽  
Xiaoying Luo ◽  
Yilin Tao ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Phenotypic Variability ◽  
Han Chinese ◽  
Tic Disorders ◽  
Trna Genes ◽  
Incomplete Penetrance ◽  
Mitochondrial Trna ◽  
Pathogenic Potential ◽  
Total Prevalence ◽  
Chinese Subjects

Abstract Aim: To conduct the clinical, genetic, and molecular characterization of 494 Han Chinese subjects with tic disorders (TD). Methods: In the present study, we performed the mutational analysis of 22 mitochondrial tRNA genes in a large cohort of 494 Han Chinese subjects with TD via Sanger sequencing. These variants were then assessed for their pathogenic potential via phylogenetic, functional, and structural analyses. Results: A total of 73 tRNA gene variants (49 known and 24 novel) on 22 tRNA genes were identified. Among these, 18 tRNA variants that were absent or present in <1% of 485 Chinese control patient samples were localized to highly conserved nucleotides, or changed the modified nucleotides, and had the potential structural to alter tRNA structure and function. These variants were thus considered to be TD-associated mutations. In total, 25 subjects carried one of these 18 putative TD-associated tRNA variants with the total prevalence of 4.96%. Limitations: The phenotypic variability and incomplete penetrance of tic disorders in pedigrees carrying these tRNA mutations suggested the involvement of modifier factors, such as nuclear encoded genes associated mitochondrion, mitochondrial haplotypes, epigenetic, and environmental factors. Conclusion: Our data provide the evidence that mitochondrial tRNA mutations are the important causes of tic disorders among Chinese population. These findings also advance current understanding regarding the clinical relevance of tRNA mutations, and will guide future studies aimed at elucidating the pathophysiology of maternal tic disorders.

scholarly journals Mutational Analysis of Mitochondrial tRNA Genes in 200 Patients with Type 2 Diabetes Mellitus

2021 ◽  
Vol Volume 14 ◽  
pp. 5719-5735
Author(s):  
Liangyan Lin ◽  
Dongdong Zhang ◽  
Qingsong Jin ◽  
Yaqin Teng ◽  
Xiaoyan Yao ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Mutational Analysis ◽  
Trna Genes ◽  
Mitochondrial Trna ◽  
Mitochondrial Trna Genes

scholarly journals RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 3020 ◽  
Author(s):  
Alan Ann Lerk Ong ◽  
Jiazi Tan ◽  
Malini Bhadra ◽  
Clément Dezanet ◽  
Kiran M. Patil ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Nucleic Acids ◽  
Splice Site ◽  
Peptide Nucleic Acids ◽  
Amino Sugar ◽  
Hairpin Structure ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Antisense Peptide ◽  
Exon 10

Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5′ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3′ arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5′ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5′ arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5′ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

scholarly journals Splice-Junction Elements and Intronic Sequences Regulate Alternative Splicing of the Drosophila Myosin Heavy Chain Gene Transcript

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 725-741 ◽  
Author(s):  
David M Standiford ◽  
Mary Beth Davis ◽  
Weitao Sun ◽  
Charles P Emerson
Keyword(s):  
Alternative Splicing ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Alternative Exon ◽  
Chain Gene ◽  
Gene Transcript ◽  
Specific Alternative ◽  
Exon 11

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exons 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5′-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

scholarly journals Screening for Mitochondrial tRNA Mutations in 318 Patients with Dilated Cardiomyopathy

2022 ◽  
Author(s):  
Yujuan Qi ◽  
Zhenhua Wu ◽  
Yaobang Bai ◽  
Yan Jiao ◽  
Peijun Li
Keyword(s):  
Dilated Cardiomyopathy ◽  
Mitochondrial Dysfunction ◽  
Copy Number ◽  
Mutational Analysis ◽  
Unknown Etiology ◽  
Trna Genes ◽  
Mtdna Copy Number ◽  
Mitochondrial Trna ◽  
Mitochondrial Functions ◽  
And Gender

Objectives: Dilated cardiomyopathy (DCM) is a complex cardiovascular disease with unknown etiology. Although nuclear genes play active roles in DCM, mitochondrial dysfunction was believed to be involved in the pathogenesis of DCM. The objective of this study is to analysis the association between mitochondrial tRNA (mt-tRNA) mutations and DCM. Material and Methods: We performed a mutational analysis of mt-tRNA genes in a cohort of 318 patients with DCM and 200 age- and gender-matched control subjects. To further assess their pathogenicity, phylogenetic analysis and mitochondrial functions including mtDNA copy number, ATP and ROS were analyzed. Results: 7 possible pathogenic mutations: MT-TL1 3302A>G, MT-TI 4295A>G, MT-TM 4435A>G, MT-TA 5655T>C, MT-TH 12201T>C, MT-TE 14692A>G and MT-TT 15927G>A were identified in DCM group but absent in controls. These mutations occurred at extremely conserved nucleotides of corresponding tRNAs, and led to the failure in tRNAs metabolism. Moreover, a significant reduction in ATP and mtDNA copy number, whereas a markedly increased in ROS level were observed in polymononuclear leukocytes (PMNs) derived from the DCM patients carrying these mt-tRNA mutations, suggesting that these mutations may cause mitochondrial dysfunction that was responsible for DCM. Conclusions: Our data indicated that mt-tRNA mutations may be the molecular basis for DCM, which shaded novel insight into the pathophysiology of DCM that was manifestated by mitochondrial dysfunction.

In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 815-815
Author(s):  
Marilyn Parra ◽  
Xiuli An ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Start Codon ◽  
Exon 2 ◽  
Mouse Tissues

Abstract Abstract 815 Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3' splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3' splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs. To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutational analysis of mitochondrial tRNA genes in patients with lung cancer

2016 ◽  
Vol 19 (2) ◽  
pp. 45-50 ◽  
Author(s):  
ZF He ◽  
LC Zheng ◽  
DY Xie ◽  
SS Yu ◽  
J Zhao
Keyword(s):  
Lung Cancer ◽  
Hot Spots ◽  
Healthy Subjects ◽  
Mutational Analysis ◽  
State Level ◽  
Trna Genes ◽  
Steady State Level ◽  
Mitochondrial Trna ◽  
Blood Samples ◽  
Mitochondrial Trna Genes

AbstractMutations in mitochondrial tRNA (mt-tRNA) genes have been found to be associated with various diseases including lung cancer. To understand the possible relationship between mtRNA mutations and lung cancer, we sequenced the 22 mt-tRNA genes from 200 lung cancer blood samples, as well as 100 healthy subjects. As a result, five mutations were identified including the tRNAAla T5655C, tRNAArg T10454C, tRNALeu(CUN) A12330G, tRNASer(UCN) T7505C and tRNAThr G15927A. These mutations were absent in the healthy subjects. These mutations and polymorphisms were localized at the highly conserved nucleotides of the corresponding mitochondrial tRNAs, which are critical for the tRNA steady state level and may result in failure in the tRNA metabolism. Moreover, through the application of the pathogenicity scoring system, we found that only the T10454C mutation should be classified as a “neutral polymorphism,” while the other mutations were regarded as “definitely pathogenic.” Taken together, our data indicate that tRNA genes are the hot-spots for pathogenic mutations associated with lung cancer. Our findings may provide valuable information for pathophysiology, management and genetic counseling of lung cancer.

scholarly journals Interplay of primary sequence and RNA secondary structure in determining 5′ splice site choice

2018 ◽  
Author(s):  
Frances Anne Tosto ◽  
Asaf Shilo ◽  
Jason W. Rausch ◽  
Stuart F. J. Le Grice ◽  
Tom Misteli
Keyword(s):  
Secondary Structure ◽  
Splice Site ◽  
Site Selection ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Premature Aging ◽  
Common Mechanism ◽  
Splice Sites ◽  
Primary Sequence ◽  
Splice Site Selection

AbstractSelective use of 5′ splice sites is a common mechanism by which pre-mRNAs are alternatively spliced. Whereas the sequence requirements of 5′ splice site choice have been well characterized, other important determinants remain poorly defined. Here we apply a combination of structural mapping by SHAPE-MaP and targeted mutational analysis in a cell-based system to comprehensively probe the interplay of primary sequence, secondary RNA structure, regulatory elements and linear splice site position to determine mechanisms of splice site choice in vivo. Using the disease-causing alternative 5′ splice site selection in LMNA in the premature aging disorder Hutchinson-Gilford Progeria Syndrome as a model system, we identify RNA secondary structural elements near the alternative 5′ splice sites. We show that splice site choice is significantly influenced by the structural context of the available splice sites. While local structure alone is not sufficient to account for splice site selection, the choice of 5′ splice sites depends on the structural stability of the 5′ splice site region which is conferred by downstream elements. In addition, relative positioning of the competing sites within the primary sequence of the pre-mRNA is a predictor of 5′ splice site usage, with the distal position favored over the proximal, regardless of sequence composition. Together, these results reveal an intricate interplay amongst RNA sequence, secondary structure and splice site position in determining 5′ splice site choice.

The ratio of ±KTS splice variants of the Wilms’ tumour suppressor protein WT1 mRNA is determined by an intronic enhancer

2008 ◽  
Vol 86 (4) ◽  
pp. 312-321 ◽  
Author(s):  
Cheng Yang ◽  
Paul J. Romaniuk
Keyword(s):  
Amino Acids ◽  
Alternative Splicing ◽  
Splice Site ◽  
Mutational Analysis ◽  
Tumour Suppressor ◽  
Wilms Tumour ◽  
Mrna Isoforms ◽  
Primary Transcript ◽  
Functional Link ◽  
Intronic Enhancer

Alternative splicing of the primary transcript of the Wilms’ tumour suppressor gene WT1 involving 2 overlapping 5′ splice sites at the end of exon 9 results in the insertion of 2 amino acids (KTS) between zinc fingers 3 and 4 of the protein. The presence or absence of these 3 amino acids has consequences for DNA binding affinity, protein–protein interactions, and subnuclear localization. Disruption of the characteristic +KTS to –KTS ratio of mRNA isoforms as a result of mutations in the +KTS splice site results in Frasier syndrome. Mutational analysis of a WT1 minigene construct was carried out to search for sequences that regulate the ±KTS alternative splicing event. A strong pyrimidine-rich intronic enhancer that increases the use of the +KTS splice site was identified. Cross-linking experiments with nuclear extracts demonstrated that this enhancer specifically binds a protein with a molecular mass of 42 ± 2 kDa. One candidate for this trans-factor is the splicing regulator TIA-1, which binds to the pyrimidine-rich enhancer in the primary transcript from the minigene construct and increases the ±KTS splicing ratio. The observation that TIA-1 and WT1 are both involved in apoptosis supports our proposal for a functional link between these proteins.

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