Human Box H/ACA Pseudouridylation Guide RNA Machinery

ABSTRACT Pseudouridine, the most abundant modified nucleoside in RNA, is synthesized by posttranscriptional isomerization of uridines. In eukaryotic RNAs, site-specific synthesis of pseudouridines is directed primarily by box H/ACA guide RNAs. In this study, we have identified 61 novel putative pseudouridylation guide RNAs by construction and characterization of a cDNA library of human box H/ACA RNAs. The majority of the new box H/ACA RNAs are predicted to direct pseudouridine synthesis in rRNAs and spliceosomal small nuclear RNAs. We can attribute RNA-directed modification to 79 of the 97 pseudouridylation sites present in the human 18S, 5.8S, and 28S rRNAs and to 11 of the 21 pseudouridines reported for the U1, U2, U4, U5, and U6 spliceosomal RNAs. We have also identified 12 novel box H/ACA RNAs which lack apparent target pseudouridines in rRNAs and small nuclear RNAs. These putative guide RNAs likely function in the pseudouridylation of some other types of cellular RNAs, suggesting that RNA-guided pseudouridylation is more general than assumed before. The genomic organization of the new box H/ACA RNA genes indicates that in human cells, all box H/ACA pseudouridylation guide RNAs are processed from introns of pre-mRNA transcripts which either encode a protein product or lack protein-coding capacity.

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Guide RNA acrobatics: positioning consecutive uridines for pseudouridylation by H/ACA pseudouridylation loops with dual guide capacity

Genes & Development ◽

10.1101/gad.349072.121 ◽

2021 ◽

Author(s):

Beáta E. Jády ◽

Amandine Ketele ◽

Dylan Moulis ◽

Tamás Kiss

Keyword(s):

Direct Synthesis ◽

Structural Flexibility ◽

Target Sequence ◽

Site Specific ◽

Guide Rna ◽

Guide Rnas ◽

Spliceosomal Rnas ◽

Target Rna ◽

Specific Synthesis ◽

Distal Stem

Site-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal “antisense” sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5′-UN-3′, including the 5′ substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (Ψ) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs.

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Characterization of the Complete Mitochondrial Genome of Fischoederius elongatus Derived from Cows in Shanghai, China

BioMed Research International ◽

10.1155/2020/7975948 ◽

2020 ◽

Vol 2020 ◽

pp. 1-6

Author(s):

Zhaoqing Han ◽

Kun Li ◽

Houqiang Luo ◽

Muhammad Shahzad ◽

Khalid Mehmood

Keyword(s):

Amino Acids ◽

Mitochondrial Genome ◽

Ribosomal Rna ◽

Parasite Species ◽

Codon Position ◽

Complete Mitochondrial Genome ◽

Nucleotide Composition ◽

Protein Coding ◽

Rna Genes

A study was conducted to reveal the characterization of the complete mitochondrial genome of Fischoederius elongatus derived from cows in Shanghai, China. Results indicated that the complete mt genome of F. elongatus was 14,288 bp and contained 12 protein-coding genes (cox1-3, nad1-6, nad4L, atp6, and cytb), 22 transfer RNA genes, and two ribosomal RNA genes (l-rRNA and s-rRNA). The overall A + T content of the mt genome was 63.83%, and the nucleotide composition was A (19.83%), C (9.75%), G (26.43%), and T (44.00%). A total of 3284 amino acids were encoded by current F. elongatus isolate mt genome, TTT (Phe) (9.84%) and TTG (Leu) (7.73%) codon were the most frequent amino acids, whereas the ACC (Thr) (0.06%), GCC (Ala) (0.09%), CTC (Leu) (0.09%), and AAC (Asn) (0.09%) codon were the least frequent ones. At the third codon position of F. elongatus mt protein genes, T (50.82%) was observed most frequently and C (5.85%) was the least one. The current results can contribute to epidemiology diagnosis, molecular identification, taxonomy, genetic, and drug development researches about this parasite species in cattle.

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Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

10.1101/2020.08.20.259762 ◽

2020 ◽

Author(s):

Xinyi Guo ◽

Hans-Hermann Wessels ◽

Alejandro Méndez-Mancilla ◽

Daniel Haro ◽

Neville E. Sanjana

Keyword(s):

Rna Virus ◽

H1n1 Influenza ◽

Model Organisms ◽

Messenger Rnas ◽

Protein Coding ◽

Guide Rna ◽

Knock Down ◽

Guide Rnas ◽

Rna Targeting ◽

Rna Design

AbstractCRISPR-Cas13 mediates robust transcript knockdown in human cells through direct RNA targeting. Compared to DNA-targeting CRISPR enzymes like Cas9, RNA targeting by Cas13 is transcript- and strand-specific: It can distinguish and specifically knock-down processed transcripts, alternatively spliced isoforms and overlapping genes, all of which frequently serve different functions. Previously, we identified optimal design rules for RfxCas13d guide RNAs (gRNAs), and developed a computational model to predict gRNA efficacy for all human protein-coding genes. However, there is a growing interest to target other types of transcripts, such as noncoding RNAs (ncRNAs) or viral RNAs, and to target transcripts in other commonly-used organisms. Here, we predicted relative Cas13-driven knock-down for gRNAs targeting messenger RNAs and ncRNAs in six model organisms (human, mouse, zebrafish, fly, nematode and flowering plants) and four abundant RNA virus families (SARS-CoV-2, HIV-1, H1N1 influenza and MERS). To allow for more flexible gRNA efficacy prediction, we also developed a web-based application to predict optimal gRNAs for any RNA target entered by the user. Given the lack of Cas13 guide design tools, we anticipate this resource will facilitate CRISPR-Cas13 RNA targeting in common model organisms, emerging viral threats to human health, and novel RNA targets.

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Characterization of the complete chloroplast genome sequence of Vitis vinifera ‘Guifeimeigui’

Scientific Journal of Genetics and Gene Therapy ◽

10.17352/sjggt.000019 ◽

2021 ◽

pp. 001-003

Author(s):

Liu Li ◽

Yang Yang ◽

Li Xiujie ◽

Li Bo

Keyword(s):

Vitis Vinifera ◽

Gc Content ◽

Single Copy ◽

Protein Coding ◽

Complete Chloroplast Genome ◽

Chloroplast Genome Sequence ◽

Cp Genome ◽

Eurasian Species ◽

Rna Genes

Vitis vinifera ‘Guifeimeigui’ is a diploid table grape, a Eurasian species. This research first reported the complete chloroplast (cp) genome of Vitis vinifera ‘Guifeimeigui’. The size of the complete cp genome is 160,928 bp and its GC content is 37.38%, including a pair of inverted repeats (26,353 bp each) separated by large (89,150 bp) and small (19,072 bp) single-copy regions. It encodes 85 genes, including 40 protein coding genes, 37 transfer RNA genes (tRNA), and 8 ribosomal RNA genes (rRNA). The Maximum Likelihood (ML) phylogenetic tree demonstrated that Vitis vinifera ‘Guifeimeigui’ is close to Vitis vinifera.

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Characterization of the mitochondrial genome ofArge bellaWei & Du sp. nov. (Hymenoptera: Argidae)

PeerJ ◽

10.7717/peerj.6131 ◽

2018 ◽

Vol 6 ◽

pp. e6131 ◽

Cited By ~ 3

Author(s):

Shiyu Du ◽

Gengyun Niu ◽

Tommi Nyman ◽

Meicai Wei

Keyword(s):

Mitochondrial Genome ◽

High Throughput Sequencing ◽

Complete Mitochondrial Genome ◽

Nucleotide Composition ◽

Sequencing Data ◽

Protein Coding ◽

High Throughput Sequencing Data ◽

Rna Genes ◽

Ancestral Type

We describeArge bellaWei & Du sp. nov., a large and beautiful species of Argidae from south China, and report its mitochondrial genome based on high-throughput sequencing data. We present the gene order, nucleotide composition of protein-coding genes (PCGs), and the secondary structures of RNA genes. The nearly complete mitochondrial genome ofA. bellahas a length of 15,576 bp and a typical set of 37 genes (22 tRNAs, 13 PCGs, and 2 rRNAs). Three tRNAs are rearranged in theA. bellamitochondrial genome as compared to the ancestral type in insects:trnMandtrnQare shuffled, whiletrnWis translocated from thetrnW-trnC-trnYcluster to a location downstream oftrnI. All PCGs are initiated by ATN codons, and terminated with TAA, TA or T as stop codons. All tRNAs have a typical cloverleaf secondary structure, except fortrnS1. H821 ofrrnSand H976 ofrrnLare redundant. A phylogenetic analysis based on mitochondrial genome sequences ofA. bella, 21 other symphytan species, two apocritan representatives, and four outgroup taxa supports the placement of Argidae as sister to the Pergidae within the symphytan superfamily Tenthredinoidea.

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Characterization of Two Complete Mitochondrial Genomes of Atkinsoniella (Hemiptera: Cicadellidae: Cicadellinae) and the Phylogenetic Implications

Insects ◽

10.3390/insects12040338 ◽

2021 ◽

Vol 12 (4) ◽

pp. 338

Author(s):

Yan Jiang ◽

Hao-Xi Li ◽

Xiao-Fei Yu ◽

Mao-Fa Yang

Keyword(s):

Stop Codon ◽

Phylogenetic Analyses ◽

Initiation Site ◽

Rrna Genes ◽

Mitochondrial Genomes ◽

Protein Coding ◽

Phylogenetic Implications ◽

Rna Genes ◽

Complete Mitochondrial Genomes

The complete mitochondrial genomes of Atkinsoniella grahami and Atkinsoniella xanthonota were sequenced. The results showed that the mitogenomes of these two species are 15,621 and 15,895 bp in length, with A+T contents of 78.6% and 78.4%, respectively. Both mitogenomes contain 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a control region (CR). For all PCGs, a standard start ATN codon (ATT, ATG, or ATA) was found at the initiation site, except for ATP8, for which translation is initiated with a TTG codon. All PCGs terminate with a complete TAA or TAG stop codon, except for COX2, which terminates with an incomplete stop codon T. All tRNAs have the typical cloverleaf secondary structure, except for trnS, which has a reduced dihydrouridine arm. Furthermore, these phylogenetic analyses were reconstructed based on 13 PCGs and two rRNA genes of 73 mitochondrial genome sequences, with both the maximum likelihood (ML) and Bayesian inference (BI) methods. The obtained mitogenome sequences in this study will promote research into the classification, population genetics, and evolution of Cicadellinae insects in the future.

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The complete mitochondrial genome of a parthenogenetic ant, Monomorium triviale (Hymenoptera: Formicidae)

10.1101/2021.03.02.433554 ◽

2021 ◽

Author(s):

Naoto Idogawa ◽

Chih-Chi Lee ◽

Chin-Cheng Scotty Yang ◽

Shigeto Dobata

Keyword(s):

Complete Mitochondrial Genome ◽

Coding Region ◽

Protein Coding ◽

Transfer Rnas ◽

Phylogenetic Characterization ◽

Reproductive Division Of Labor ◽

Reproductive Division ◽

Rna Genes ◽

Generation Sequencing

ABSTRACTMonomorium is one of the most species-rich yet taxonomically problematic ant genera. An East Asian species, M. triviale Wheeler, W.M., 1906, reproduces by obligate thelytokous parthenogenesis and performs strict reproductive division of labor. We sequenced the M. triviale mitogenome using next-generation sequencing methods. The circular mitogenome of M. triviale was 16,290 bp in length, consisting of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs, and a single non-coding region of 568 bp. The base composition was AT-biased (82%). Gene order rearrangements were detected and likely to be unique to genus Monomorium. We announce the M. triviale mitogenome as additional genomic resources for phylogenetic characterization of Monomorium and comparative genomics of parthenogenetic ant species.

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Characterization of the mitochondrial genome of the pathogenic fungus Scytalidium auriculariicola (Leotiomycetes) and insights into its phylogenetics

Scientific Reports ◽

10.1038/s41598-019-53941-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Cheng Chen ◽

Qiang Li ◽

Rongtao Fu ◽

Jian Wang ◽

Chuan Xiong ◽

...

Keyword(s):

Mitochondrial Genome ◽

Pathogenic Fungus ◽

Phylogenetic Analyses ◽

Single Gene ◽

Protein Coding ◽

Next Generation Sequencing Technology ◽

Protein Coding Genes ◽

Rna Genes ◽

Generation Sequencing

AbstractScytalidium auriculariicola is the causative pathogen of slippery scar disease in the cultivated cloud ear fungus, Auricularia polytricha. In the present study, the mitogenome of S. auriculariicola was sequenced and assembled by next-generation sequencing technology. The circular mitogenome is 96,857 bp long and contains 56 protein-coding genes, 2 ribosomal RNA genes, and 30 transfer RNA genes (tRNAs). The high frequency of A and T used in codons contributed to the high AT content (73.70%) of the S. auriculariicola mitogenome. Comparative analysis indicated that the base composition and the number of introns and protein-coding genes in the S. auriculariicola mitogenome varied from that of other Leotiomycetes mitogenomes, including a uniquely positive AT skew. Five distinct groups were found in the gene arrangements of Leotiomycetes. Phylogenetic analyses based on combined gene datasets (15 protein-coding genes) yielded well-supported (BPP = 1) topologies. A single-gene phylogenetic tree indicated that the nad4 gene may be useful as a molecular marker to analyze the phylogenetic relationships of Leotiomycetes species. This study is the first report on the mitochondrial genome of the genus Scytalidium, and it will contribute to our understanding of the population genetics and evolution of S. auriculariicola and related species.

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Principles for rational Cas13d guide design

10.1101/2019.12.27.889089 ◽

2019 ◽

Cited By ~ 4

Author(s):

Hans-Hermann Wessels ◽

Alejandro Méndez-Mancilla ◽

Xinyi Guo ◽

Mateusz Legut ◽

Zharko Daniloski ◽

...

Keyword(s):

Fluorescent Protein ◽

Perfect Match ◽

Nuclease Activity ◽

Target Site ◽

Surface Proteins ◽

Protein Coding ◽

Guide Rna ◽

Knock Down ◽

Guide Rnas ◽

Green Fluorescent

AbstractType VI CRISPR enzymes have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allow for specific and robust target gene knock-down without altering the genome. However, we currently lack information about optimal Cas13 guide RNA designs for high target RNA knock-down efficacy. To close this gap, we conducted four massively-parallel Cas13 screens targeting the mRNA of a destabilized green fluorescent protein (GFP) transgene and CD46, CD55 and CD71 cell surface proteins in human cells. In total, we measured the activity of 24,460 guide RNA including 6,469 perfect match guide RNAs and a diverse set of guide RNA variants and permutations with mismatches relative to the target sequences.We find that guide RNAs show high diversity in knock-down efficiency driven by crRNA-specific features as well as target site context. Moreover, while single mismatches generally reduce knock-down to a modest degree, we identify a critical region spanning spacer nucleotides 15 – 21 that is largely intolerant to target site mismatches. We developed a computational model to identify guide RNAs with high knock-down efficacy. We confirmed the model’s generalizability across a large number of endogenous target mRNAs and show that Cas13 can be used in forward genetic pooled CRISPR-screens to identify essential genes. Using this model, we provide a resource of optimized Cas13 guide RNAs to target all protein-coding transcripts in the human genome, enabling transcriptome-wide forward genetic screens.

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CRISPRO Identifies Functional Protein Coding Sequences Based on Genome Editing Dense Mutagenesis

10.1101/326504 ◽

2018 ◽

Cited By ~ 1

Author(s):

Vivien A. C. Schoonenberg ◽

Mitchel A. Cole ◽

Qiuming Yao ◽

Claudio Macias-Treviño ◽

Falak Sher ◽

...

Keyword(s):

Machine Learning ◽

Computational Pipeline ◽

Functional Protein ◽

Protein Coding ◽

Coding Sequences ◽

Guide Rna ◽

Guide Rnas ◽

Functional Scores ◽

Parallel Evaluation

AbstractCRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets we have developed CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genome, transcript, and protein coordinates and structure. No available tool has similar functionality. The ensuing genotype-phenotype linear and 3D maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro.

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