Sequence features around cleavage sites are highly conserved among different species and a critical determinant for RNA cleavage position across eukaryotes

ABSTRACTRNA degradation is critical for control of gene expression, and endonucleolytic cleavage– dependent RNA degradation is conserved among eukaryotes. Some cleavage sites are secondarily capped in the cytoplasm and identified using the CAGE method. Although uncapped cleavage sites are widespread in eukaryotes, comparatively little information has been obtained about these sites using CAGE-based degradome analysis. Previously, we developed the truncated RNA-end sequencing (TREseq) method in plant species and used it to acquire comprehensive information about uncapped cleavage sites; we observed G-rich sequences near cleavage sites. However, it remains unclear whether this finding is general to other eukaryotes. In this study, we conducted TREseq analyses in fruit flies (Drosophila melanogaster) and budding yeast (Saccharomyces cerevisiae). The results revealed specific sequence features related to RNA cleavage in D. melanogaster and S. cerevisiae that were similar to sequence patterns in Arabidopsis thaliana. Although previous studies suggest that ribosome movements are important for determining cleavage position, feature selection using a random forest classifier showed that sequences around cleavage sites were major determinant for cleaved or uncleaved sites. Together, our results suggest that sequence features around cleavage sites are critical for determining cleavage position, and that sequence-specific endonucleolytic cleavage–dependent RNA degradation is highly conserved across eukaryotes.

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Feature selection for RNA cleavage efficiency at specific sites using the LASSO regression model in Arabidopsis thaliana

BMC Bioinformatics ◽

10.1186/s12859-021-04291-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Daishin Ueno ◽

Harunori Kawabe ◽

Shotaro Yamasaki ◽

Taku Demura ◽

Ko Kato

Keyword(s):

Feature Selection ◽

Regression Model ◽

Rna Degradation ◽

Rna Cleavage ◽

Sequence Information ◽

Cleavage Sites ◽

Lasso Regression ◽

Translation Process ◽

Cleavage Efficiency ◽

Exogenous Genes

Abstract Background RNA degradation is important for the regulation of gene expression. Despite the identification of proteins and sequences related to deadenylation-dependent RNA degradation in plants, endonucleolytic cleavage-dependent RNA degradation has not been studied in detail. Here, we developed truncated RNA end sequencing in Arabidopsis thaliana to identify cleavage sites and evaluate the efficiency of cleavage at each site. Although several features are related to RNA cleavage efficiency, the effect of each feature on cleavage efficiency has not been evaluated by considering multiple putative determinants in A. thaliana. Results Cleavage site information was acquired from a previous study, and cleavage efficiency at the site level (CSsite value), which indicates the number of reads at each cleavage site normalized to RNA abundance, was calculated. To identify features related to cleavage efficiency at the site level, multiple putative determinants (features) were used to perform feature selection using the Least Absolute Shrinkage and Selection Operator (LASSO) regression model. The results indicated that whole RNA features were important for the CSsite value, in addition to features around cleavage sites. Whole RNA features related to the translation process and nucleotide frequency around cleavage sites were major determinants of cleavage efficiency. The results were verified in a model constructed using only sequence features, which showed that the prediction accuracy was similar to that determined using all features including the translation process, suggesting that cleavage efficiency can be predicted using only sequence information. The LASSO regression model was validated in exogenous genes, which showed that the model constructed using only sequence information can predict cleavage efficiency in both endogenous and exogenous genes. Conclusions Feature selection using the LASSO regression model in A. thaliana identified 155 features. Correlation coefficients revealed that whole RNA features are important for determining cleavage efficiency in addition to features around the cleavage sites. The LASSO regression model can predict cleavage efficiency in endogenous and exogenous genes using only sequence information. The model revealed the significance of the effect of multiple determinants on cleavage efficiency, suggesting that sequence features are important for RNA degradation mechanisms in A. thaliana.

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Defining the transcriptional and post-transcriptional landscapes ofMycobacterium smegmatisin aerobic growth and oxygen limitation

10.1101/423392 ◽

2018 ◽

Author(s):

M. Carla Martini ◽

Ying Zhou ◽

Huaming Sun ◽

Scarlet S. Shell

Keyword(s):

Gene Expression ◽

Model Organism ◽

Rna Cleavage ◽

Binding Motif ◽

Sequence Motif ◽

Cleavage Sites ◽

Low Oxygen ◽

Phase Growth ◽

Genomic Context ◽

Control Of Gene Expression

AbstractThe ability ofMycobacterium tuberculosisto infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5’-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organismMycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that likeM. tuberculosis,M. smegmatiscan initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Our results show thatM. smegmatisandM. tuberculosisshare a large number of similarities at the transcriptomic level, suggesting that similar regulatory mechanisms govern both species.

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Toxins from TA system of Helicobacter pylori and insight into mRNase activity

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314091712 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C828-C828

Author(s):

Chinar Pathak ◽

Hookang Im ◽

Sun-bok Jang ◽

Yeon-Jin Yang ◽

Hye-Jin Yoon ◽

...

Keyword(s):

Helicobacter Pylori ◽

Active Site ◽

Isothermal Calorimetry ◽

Rna Degradation ◽

Base Catalysis ◽

Homology Search ◽

Specific Sequence ◽

General Base ◽

Sequence Recognition ◽

Calorimetric Studies

The toxin-antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. The HP0892-HP0893 and HP0894-HP0895 toxin-antitoxin systems are the only two known TA systems belonging to Helicobacter pylori. In both of these TA systems, the antitoxin binds and inhibits the toxin and regulates the transcription of the TA operon. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remains unclear. Therefore, here an attempt was made to shed some light on the mechanism behind the TA system of HP0892-HP0893 and HP0894-HP0895. Here, we present the crystal structures of apo- and copper-bound HP0894 at 1.28 Å and 1.89 Å, respectively, and the crystal structure of the zinc-bound HP0892 toxin at 1.8 Å resolution. Reorientation of residues involving the mRNase active site was shown. Through the combined approach of structural analysis along with isothermal calorimetry studies and structural homology search, the amino acids involved in mRNase active site were monitored. In the mRNase active site of HP0894 toxin, His84 acts as a catalytic residue and reorients itself acting as a general acid in an acid-base catalysis reaction, while His47 and His60 stabilize the transition state. Glu58 acts as a general base, and substrate reorientation is caused by Phe88. In the mRNase active site of HP0892 toxin, the most catalytically important residue, His86, reorients itself to exhibit RNase activity while Glu58 acts as a general base. His47 and His60 are considered to be involved in enzymatic activity. Glu58 and Asp64 are involved in substrate binding and specific sequence recognition. The mutational constructs were used for isothermal calorimetric studies to analyze the effect of catalytic residues.

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Functional expression of ribozymes in Apicomplexa: Towards exogenous control of gene expression by inducible RNA-cleavage

International Journal for Parasitology ◽

10.1016/j.ijpara.2007.10.015 ◽

2008 ◽

Vol 38 (6) ◽

pp. 673-681 ◽

Cited By ~ 21

Author(s):

Carolina Agop-Nersesian ◽

Judith Pfahler ◽

Michael Lanzer ◽

Markus Meissner

Keyword(s):

Gene Expression ◽

Functional Expression ◽

Rna Cleavage ◽

Control Of Gene Expression ◽

Exogenous Control

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Sequence Features of Drosha and Dicer Cleavage Sites Affect the Complexity of IsomiRs

International Journal of Molecular Sciences ◽

10.3390/ijms16048110 ◽

2015 ◽

Vol 16 (12) ◽

pp. 8110-8127 ◽

Cited By ~ 33

Author(s):

Julia Starega-Roslan ◽

Tomasz Witkos ◽

Paulina Galka-Marciniak ◽

Wlodzimierz Krzyzosiak

Keyword(s):

Cleavage Sites ◽

Sequence Features ◽

Dicer Cleavage

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Deconvolving sequence features that discriminate between overlapping regulatory annotations

10.1101/100511 ◽

2017 ◽

Author(s):

Akshay Kakumanu ◽

Silvia Velasco ◽

Esteban Mazzoni ◽

Shaun Mahony

Keyword(s):

Embryonic Stem ◽

Cell Types ◽

The Novel ◽

Cell Type ◽

Dynamic Binding ◽

Specific Sequence ◽

Sequence Features ◽

Binding Behavior ◽

Distinct Sequence ◽

Regulatory Sites

AbstractGenomic loci with regulatory potential can be identified and annotated with various properties. For example, genomic sites may be annotated as being bound by a given transcription factor (TF) in one or more cell types. The same sites may be further labeled as being proximal or distal to known promoters. Given such a collection of labeled sites, it is natural to ask what sequence features are associated with each annotation label. However, discovering such label-specific sequence features is often confounded by overlaps between annotation labels; e.g. if regulatory sites specific to a given cell type are also more likely to be promoter-proximal, it is difficult to assess whether motifs identified in that set of sites are associated with the cell type or associated with promoters. In order to meet this challenge, we developed SeqUnwinder, a principled approach to deconvolving interpretable discriminative sequence features associated with overlapping annotation labels. We demonstrate the novel analysis abilities of SeqUnwinder using three examples. Firstly, we show SeqUnwinder’s ability to unravel sequence features associated with the dynamic binding behavior of TFs during motor neuron programming from features associated with chromatin state in the initial embryonic stem cells. Secondly, we characterize distinct sequence properties of multi-condition and cell-specific TF binding sites after controlling for uneven associations with promoter proximity. Finally, we demonstrate the scalability of SeqUnwinder to discover cell-specific sequence features from over one hundred thousand genomic loci that display DNase I hypersensitivity in one or more ENCODE cell lines.Availabilityhttps://github.com/seqcode/sequnwinder

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The domain architecture of JBP1 suggests synergy between J-base DNA binding and thymidine hydroxylase activity

10.1101/502252 ◽

2018 ◽

Cited By ~ 1

Author(s):

Athanassios Adamopoulos ◽

Tatjana Heidebrecht ◽

Jeroen Roosendaal ◽

Wouter G Touw ◽

Isabelle Phan ◽

...

Keyword(s):

Dna Binding ◽

Domain Architecture ◽

Three Dimensional ◽

Specific Sequence ◽

X Ray ◽

Tet Proteins ◽

X Ray Scattering ◽

Sequence Patterns ◽

Three Dimensional Models

JBP1 (J-DNA Binding Protein 1) contributes to biosynthesis and maintenance of base J (β-D-glucosyl-hydroxymethyluracil), a modification of thymidine (T) confined to pathogenic protozoa. JBP1 has two known functional domains: an N-terminal thymidine hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins; and a J-DNA binding domain (JDBD) that resides in the middle of JBP1. Here we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered domain. This Δ-JDBD domain retains thymidine hydroxylation activity in vitro, but displays a fifteen-fold lower apparent rate of hydroxylation compared to JBP1. Small Angle X-ray Scattering (SAXS) experiments on JBP1 and JDBD in the presence and absence of J-DNA, and on Δ-JDBD, allowed us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate hydroxylation a T 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module in the Δ-JDBD scaffold during evolution provided a mechanism to synergize between J recognition and T hydroxylation, ensuring inheritance of J in specific sequence patterns following DNA replication.

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RNase E Enzymes from Rhodobacter capsulatus and Escherichia coli Differ in Context- and Sequence-Dependent In Vivo Cleavage within the Polycistronicpuf mRNA

Journal of Bacteriology ◽

10.1128/jb.181.24.7621-7625.1999 ◽

1999 ◽

Vol 181 (24) ◽

pp. 7621-7625 ◽

Cited By ~ 7

Author(s):

Claudia Heck ◽

Elena Evguenieva-Hackenberg ◽

Angelika Balzer ◽

Gabriele Klug

Keyword(s):

Escherichia Coli ◽

Rhodobacter Capsulatus ◽

Cleavage Sites ◽

Rnase E ◽

Endonucleolytic Cleavage ◽

Sequence Dependent

ABSTRACT The 5′ pufQ mRNA segment and the pufLMXmRNA segment of Rhodobacter capsulatus exhibit different stabilities. Degradation of both mRNA segments is initiated by RNase E-mediated endonucleolytic cleavage. While RhodobacterRNase E does not discriminate between the different sequences present around the cleavage sites within pufQ and pufL,Escherichia coli RNase E shows preference for the sequence harboring more A and U residues.

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