scholarly journals Analysis of Emerging Variants in Structured Regions of the SARS-CoV-2 Genome

2021 ◽  
Vol 17 ◽  
pp. 117693432110141
Author(s):  
Sean P Ryder ◽  
Brittany R Morgan ◽  
Peren Coskun ◽  
Katianna Antkowiak ◽  
Francesca Massi
Keyword(s):  
High Throughput Sequencing ◽  
Hypervariable Region ◽  
Regulatory Elements ◽  
Rna Structures ◽  
Stem Loop ◽  
Selfish Genetic Element ◽  
Functional Relevance ◽  
The Stability ◽  
Genome Heterogeneity ◽  
A Minor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two sets of SARS-CoV-2 genomic sequences to identify emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, 20 variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5ʹ untranslated region (UTR), including a group of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-ss molecular switch in the 3ʹUTR. Finally, 5 variants destabilize structured elements within the 3ʹUTR hypervariable region, including the S2M (stem loop 2 m) selfish genetic element, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. Our analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences.

scholarly journals Analysis of Rapidly Emerging Variants in Structured Regions of the SARS-CoV-2 Genome

2020 ◽  
Author(s):  
Sean P. Ryder ◽  
Brittany R. Morgan ◽  
Francesca Massi
Keyword(s):  
High Throughput Sequencing ◽  
Hypervariable Region ◽  
Regulatory Elements ◽  
Regulatory Rna ◽  
Rna Structures ◽  
Stem Loop ◽  
Functional Relevance ◽  
The Stability ◽  
Genome Heterogeneity ◽  
A Minor

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two cohorts of SARS-CoV-2 genomic sequences to identify rapidly emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, twenty variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5’UTR, including a set of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-stable molecular switch in the 3’UTR. Finally, five variants destabilize structured elements within the 3’UTR hypervariable region, including the S2M stem loop, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. This analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences, as rapidly emerging variations in these regions could lead to drug resistance.

scholarly journals Massively parallel analysis of human 3′ UTRs reveals that AU-rich element length and registration predict mRNA destabilization

2021 ◽  
Author(s):  
David A Siegel ◽  
Olivier Le Tonqueze ◽  
Anne Biton ◽  
Noah Zaitlen ◽  
David J Erle
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Stem Loop ◽  
Mrna Destabilization ◽  
The Stability ◽  
Massively Parallel Reporter Assay ◽  
Simple Sequence

Abstract AU-rich elements (AREs) are 3′ UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3′ UTR sequences that conform to these motifs affect their function. Here we use functional annotation of sequences from 3′ UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3′ UTR sequence fragments from 4,653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3′ UTRs.

scholarly journals Massively Parallel Analysis of Human 3′ UTRs Reveals that AU-Rich Element Length and Registration Predict mRNA Destabilization

2020 ◽  
Author(s):  
David A. Siegel ◽  
Olivier Le Tonqueze ◽  
Anne Biton ◽  
Noah Zaitlen ◽  
David J. Erle
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Regulatory Elements ◽  
Jurkat Cells ◽  
Massively Parallel ◽  
Stem Loop ◽  
New Approach ◽  
Mrna Destabilization ◽  
The Stability ◽  
Massively Parallel Reporter Assay

AbstractAU-rich elements (AREs) are 3′ UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3′ UTR sequences that conform to these motifs affect their function. Here we use functional annotation of sequences from 3′ UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3′ UTR sequence fragments from 4,653 transcripts on gene expression and mRNA stability. The library included 9,142 AREs, and incorporated a set of fragments bearing mutations in each ARE. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Our new approach explains 64±13% of the contribution of AREs to the stability of human 3′ UTRs in Jurkat cells and predicts ARE activity in an unrelated cell type. Finally, to investigate the advantages of our general experimental design for annotating 3′ UTR elements we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3′ UTRs containing functional motifs.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM

2020 ◽  
Author(s):  
MAK Williams ◽  
V Cornuault ◽  
AH Irani ◽  
VV Symonds ◽  
J Malmström ◽  
...  
Keyword(s):  
Average Length ◽  
American Chemical Society ◽  
Md Simulations ◽  
Chemical Society ◽  
Side Chains ◽  
Rhamnogalacturonan I ◽  
Afm Imaging ◽  
Minor Population ◽  
The Stability ◽  
A Minor

© 2020 American Chemical Society. Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 μm long was observed.

Extraction of strontium and barium salts by the nitrobenzene solution of dicarbolide in the presence of glymes

1985 ◽  
Vol 50 (3) ◽  
pp. 581-599 ◽  
Author(s):  
Petr Vaňura ◽  
Emanuel Makrlík
Keyword(s):  
Stability Constants ◽  
Organic Phase ◽  
Equilibrium Constants ◽  
Minor Role ◽  
Nitrobenzene Solution ◽  
Ligand Structure ◽  
Stability Constants Of Complexes ◽  
Separation Factors ◽  
The Stability ◽  
A Minor

Extraction of microamounts of Sr2+ and Ba2+ (henceforth M2+) from the aqueous solutions of perchloric acid (0.0125-1.02 mol/l) by means of the nitrobenzene solutions of dicarbolide (0.004-0.05 mol/l of H+{Co(C2B9H11)2}-) was studied in the presence of monoglyme (only Ba2+), diglyme, triglyme, and tetraglyme (CH3O-(CH2-CH2O)nCH3, where n = 1, 2, 3, 4). The distribution of glyme betweeen the aqueous and organic phases, the extraction of the protonized glyme molecule HL+ together with the extraction of M2+ ion and of the glyme complex with the M2+ ion, i.e., ML2+ (where L is the molecule of glyme), were found to be the dominating reactions in the systems under study. In the systems with tri- and tetraglymes the extraction of H+ and M2+ ions solvated with two glyme molecules, i.e., the formation of HL2+ and ML22+ species, can probably play a minor role. The values of the respective equilibrium constants, of the stability constants of complexes formed in the organic phase, and the theoretical separation factors αBa/Sr were determined. The effect of the ligand structure on the values of extraction and stability constants in the organic phase is discussed.

scholarly journals MAC5, an RNA-binding protein, protects pri-miRNAs from SERRATE-dependent exoribonuclease activities

2020 ◽  
Vol 117 (38) ◽  
pp. 23982-23990 ◽  
Author(s):  
Shengjun Li ◽  
Mu Li ◽  
Kan Liu ◽  
Huimin Zhang ◽  
Shuxin Zhang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Protein ◽  
Dual Role ◽  
Mirna Biogenesis ◽  
Core Component ◽  
Stem Loop ◽  
Loop Region ◽  
Nuclear Rna ◽  
Efficient Processing ◽  
The Stability

MAC5 is a component of the conserved MOS4-associated complex. It plays critical roles in development and immunity. Here we report that MAC5 is required for microRNA (miRNA) biogenesis. MAC5 interacts with Serrate (SE), which is a core component of the microprocessor that processes primary miRNA transcripts (pri-miRNAs) into miRNAs and binds the stem-loop region of pri-miRNAs. MAC5 is essential for both the efficient processing and the stability of pri-miRNAs. Interestingly, the reduction of pri-miRNA levels inmac5is partially caused by XRN2/XRN3, the nuclear-localized 5′-to-3′ exoribonucleases, and depends on SE. These results reveal that MAC5 plays a dual role in promoting pri-miRNA processing and stability through its interaction with SE and/or pri-miRNAs. This study also uncovers that pri-miRNAs need to be protected from nuclear RNA decay machinery, which is connected to the microprocessor.

scholarly journals Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (9) ◽  
pp. 6337-6349 ◽  
Author(s):  
S E Wells ◽  
M Ares
Keyword(s):  
Synthetic Lethality ◽  
Small Nuclear Rna ◽  
Rna Structures ◽  
Stem Loop ◽  
U2 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

A global phylogenetic analysis of Japanese tonsil-derived Epstein–Barr virus strains using viral whole-genome cloning and long-read sequencing

2021 ◽  
Author(s):  
Misako Yajima ◽  
Risako Kakuta ◽  
Yutaro Saito ◽  
Shiori Kitaya ◽  
Atsushi Toyoda ◽  
...  
Keyword(s):  
Viral Genome ◽  
Epstein Barr Virus ◽  
High Throughput Sequencing ◽  
Phylogenetic Analyses ◽  
Regional Distribution ◽  
Genome Sequences ◽  
Barr Virus ◽  
Endemic Areas ◽  
Epstein Barr ◽  
Genome Heterogeneity

Epstein–Barr virus (EBV) establishes lifelong latent infection in the majority of healthy individuals, while it is a causative agent for various diseases, including some malignancies. Recent high-throughput sequencing results indicate that there are substantial levels of viral genome heterogeneity among different EBV strains. However, the extent of EBV strain variation among asymptomatically infected individuals remains elusive. Here, we present a streamlined experimental strategy to clone and sequence EBV genomes derived from human tonsillar tissues, which are the reservoirs of asymptomatic EBV infection. Complete EBV genome sequences, including those of repetitive regions, were determined for seven tonsil-derived EBV strains. Phylogenetic analyses based on the whole viral genome sequences of worldwide non-tumour-derived EBV strains revealed that Asian EBV strains could be divided into several distinct subgroups. EBV strains derived from nasopharyngeal carcinoma-endemic areas constitute different subgroups from a subgroup of EBV strains from non-endemic areas, including Japan. The results could be consistent with biased regional distribution of EBV-associated diseases depending on the different EBV strains colonizing different regions in Asian countries.

scholarly journals Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens

2021 ◽  
Vol 7 (27) ◽  
pp. eabf5733
Author(s):  
Rui Lopes ◽  
Kathleen Sprouffske ◽  
Caibin Sheng ◽  
Esther C. H. Uijttewaal ◽  
Adriana Emma Wesdorp ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Regulatory Networks ◽  
Essential Role ◽  
Regulatory Elements ◽  
Transcriptional Regulatory Networks ◽  
Transcriptional Regulatory Elements ◽  
Transcriptional Regulatory ◽  
Functional Relevance ◽  
Genome Scale ◽  
Transcription Program

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

scholarly journals Transcripts repressed at the stop of phloem unloading highlight the energy efficiency of sugar import in the ripening V. vinifera fruit.

2021 ◽  
Author(s):  
Stefania Savoi ◽  
Laurent Torregrosa ◽  
Charles Romieu
Keyword(s):  
Cell Wall ◽  
Regulatory Elements ◽  
Solute Concentration ◽  
Growth Patterns ◽  
Individual Growth ◽  
Sugar Accumulation ◽  
Minor Role ◽  
Phloem Unloading ◽  
Sugar Transporters ◽  
A Minor

AbstractTranscriptomic changes at the cessation of sugar accumulation in the pericarp of Vitis vinifera were addressed on single berries re-synchronized according to their individual growth patterns. The net rates of water, sugars and K+ accumulation inferred from individual growth and solute concentration confirmed that these inflows stopped simultaneously in the ripe berry, while the small amount of malic acid remaining at this stage was still being oxidized at a low rate. Resynchronized individual berries displayed negligible variations in gene expression among triplicates. RNA-Seq studies revealed sharp reprogramming of cell wall enzymes and structural proteins, associated with an 80% repression of specific sugar transporters and aquaporins on the plasma or tonoplast membranes, at the stop of phloem unloading in the three genotypes and two environments investigated. The prevalence of SWEET transporters suggests that electrogenic transporters would just play a minor role on the plasma membrane of SE/CC complex, and the one of the flesh, while sucrose/H+ exchangers dominate on its tonoplast. Cis-regulatory elements present in their promoters allowed to sort these transporters in different groups, also including specific TIPs and PIPs paralogs, and cohorts of cell wall related genes. These results lead us to propose which structural, developmental and energy adaptations would give this fruit such a power of attraction for water and photoassimilates.

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