Mutational Cold Spots Research Articles

SARS-CoV-2 main protease (M-pro) mutational profiling: An insight into mutation coldspots

SARS-CoV-2 and the COVID-19 pandemic have marked a milestone in the history of scientific research worldwide. To ensure that treatments are successful in the mid-long term, it is crucial to characterize SARS-CoV-2 mutations, as they might lead to viral resistance. Data from >5,700,000 SARS-CoV-2 genomes available at GISAID was used to report SARS-CoV-2 mutations. Given the pivotal role of its main protease (M-pro) in virus replication, a detailed analysis of SARS-CoV-2 M-pro mutations was conducted, with particular attention to mutation-resistant residues or mutation coldspots, defined as those residues that have mutated in five or fewer genomes. 32 mutation coldspots were identified, most of which mediate interprotomer interactions or funneling interaction networks from the substrate-binding site towards the dimerization surface and vice versa. Besides, mutation coldspots were virtually conserved in all main proteases from other CoVs. Our results provide valuable information about key residues to M-pro structure that could be useful in rational target-directed drug design and establish a solid groundwork based on mutation analyses for the inhibition of M-pro dimerization, with a potential applicability to future coronavirus outbreaks.

Genomic and proteomic mutation landscapes of SARS-CoV-2.

The ongoing pandemic caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2), affects thousands of people every day worldwide. Hence, drugs and vaccines effective against all variants of SARS‐CoV‐2 are crucial today. Viral genome mutations exist commonly which may impact the encoded proteins, possibly resulting to varied effectivity of detection tools and disease treatment. Thus, this study surveyed the SARS‐CoV‐2 genome and proteome and evaluated its mutation characteristics. Phylogenetic analyses of SARS‐CoV‐2 genes and proteins show three major clades and one minor clade (P6810S; ORF1ab). The overall frequency and densities of mutations in the genes and proteins of SARS‐CoV‐2 were observed. Nucleocapsid exhibited the highest mutation density among the structural proteins while the spike D614G was the most common, occurring mostly in genomes outside China and United States. ORF8 protein had the highest mutation density across all geographical areas. Moreover, mutation hotspots neighboring and at the catalytic site of RNA‐dependent RNA polymerase were found that might challenge the binding and effectivity of remdesivir. Mutation coldspots may present as conserved diagnostic and therapeutic targets were found in ORF7b, ORF9b, and ORF14. These findings suggest that the virion's genotype and phenotype in a specific population should be considered in developing diagnostic tools and treatment options.

Conserved Noncoding Elements Influence the Transposable Element Landscape in Drosophila.

Highly conserved noncoding elements (CNEs) constitute a significant proportion of the genomes of multicellular eukaryotes. The function of most CNEs remains elusive, but growing evidence indicates they are under some form of purifying selection. Noncoding regions in many species also harbor large numbers of transposable element (TE) insertions, which are typically lineage specific and depleted in exons because of their deleterious effects on gene function or expression. However, it is currently unknown whether the landscape of TE insertions in noncoding regions is random or influenced by purifying selection on CNEs. Here, we combine comparative and population genomic data in Drosophila melanogaster to show that the abundance of TE insertions in intronic and intergenic CNEs is reduced relative to random expectation, supporting the idea that selective constraints on CNEs eliminate a proportion of TE insertions in noncoding regions. However, we find no evidence for differences in the allele frequency spectra for polymorphic TE insertions in CNEs versus those in unconstrained spacer regions, suggesting that the distribution of fitness effects acting on observable TE insertions is similar across different functional compartments in noncoding DNA. Our results provide evidence that selective constraints on CNEs contribute to shaping the landscape of TE insertion in eukaryotic genomes, and provide further evidence that CNEs are indeed functionally constrained and not simply mutational cold spots.

Highly constrained intergenic Drosophila ultraconserved elements are candidate ncRNAs.

Eukaryotes contain short (∼80–200 bp) regions that have few or no substitutions among species that represent hundreds of millions of years of evolutionary divergence. These ultraconserved elements (UCEs) are candidates for containing essential functions, but their biological roles remain largely unknown. Here, we report the discovery and characterization of UCEs from 12 sequenced Drosophila species. We identified 98 elements ≥80 bp long with very high conservation across the Drosophila phylogeny. Population genetic analyses reveal that these UCEs are not present in mutational cold spots. Instead we infer that they experience a level of selective constraint almost 10-fold higher compared with missense mutations in protein-coding sequences, which is substantially higher than that observed previously for human UCEs. About one-half of these Drosophila UCEs overlap the transcribed portion of genes, with many of those that are within coding sequences likely to correspond to sites of ADAR-dependent RNA editing. For the remaining UCEs that are in nongenic regions, we find that many are potentially capable of forming RNA secondary structures. Among ten chosen for further analysis, we discovered that the majority are transcribed in multiple tissues of Drosophila melanogaster. We conclude that Drosophila species are rich with UCEs and that many of them may correspond to novel noncoding RNAs.

The mystery of extreme non-coding conservation

Regions of several dozen to several hundred base pairs of extreme conservation have been found in non-coding regions in all metazoan genomes. The distribution of these elements within and across genomes has suggested that many have roles as transcriptional regulatory elements in multi-cellular organization, differentiation and development. Currently, there is no known mechanism or function that would account for this level of conservation at the observed evolutionary distances. Previous studies have found that, while these regions are under strong purifying selection, and not mutational coldspots, deletion of entire regions in mice does not necessarily lead to identifiable changes in phenotype during development. These opposing findings lead to several questions regarding their functional importance and why they are under strong selection in the first place. In this perspective, we discuss the methods and techniques used in identifying and dissecting these regions, their observed patterns of conservation, and review the current hypotheses on their functional significance.

Positive and Negative Selection in Murine Ultraconserved Noncoding Elements

There are many more selectively constrained noncoding than coding nucleotides in the mammalian genome, but most mammalian noncoding DNA is subject to weak selection, on average. One of the most striking discoveries to have emerged from comparisons among mammalian genomes is the hundreds of noncoding elements of more than 200 bp in length that show absolute conservation among mammalian orders. These elements represent the tip of the iceberg of a much larger class of conserved noncoding elements (CNEs). Much evidence suggests that CNEs are selectively constrained and not mutational cold-spots, and there is evidence that some CNEs play a role in the regulation of development. Here, we quantify negative and positive selection acting in murine CNEs by analyzing within-species nucleotide variation and between-species divergence of CNEs that we identified using a phylogenetically independent comparison. The distribution of fitness effects of new mutations in CNEs, inferred from within-species polymorphism, suggests that CNEs receive a higher number of strongly selected deleterious mutations and many fewer nearly neutral mutations than amino acid sites of protein-coding genes or regulatory elements close to genes. However, we also show that CNEs experience a far higher proportion of adaptive substitutions than any known category of genomic sites in murids. The absolute rate of adaptation of CNEs is similar to that of amino acid sites of proteins. This result suggests that there is widespread adaptation in mammalian conserved noncoding DNA elements, some of which have been implicated in the regulation of crucially important processes, including development.

Targets of somatic hypermutation within immunoglobulin light chain genes in zebrafish

In mammals, somatic hypermutation (SHM) of immunoglobulin (Ig) genes is critical for the generation of high-affinity antibodies and effective immune responses. Knowledge of sequence-specific biases in the targeting of somatic mutations can be useful for studies aimed at understanding antibody repertoires produced in response to infections, B-cell neoplasms, or autoimmune disease. To evaluate potential nucleotide targets of somatic mutation in zebrafish (Danio rerio), an enriched IgL cDNA library was constructed and > 250 randomly selected clones were sequenced and analysed. In total, 55 unique VJ-C sequences were identified encoding a total of 125 mutations. Mutations were most prevalent in V(L) with a bias towards single base transitions and increased mutation in the complementarity-determining regions (CDRs). Overall, mutations were overrepresented at WRCH/DGYW motifs suggestive of activation-induced cytidine deaminase (AID) targeting which is common in mice and humans. In contrast to mammalian models, N and P addition was not observed and mutations at AID hotspots were largely restricted to palindromic WRCH/DGYW motifs. Mutability indexes for di- and trinucleotide combinations confirmed C/G targets within WRCH/DGYW motifs to be statistically significant mutational hotspots and showed trinucleotides ATC and ATG to be mutation coldspots. Additive mutations in VJ-C sequences revealed patterns of clonal expansion consistent with affinity maturation responses seen in higher vertebrates. Taken together, the data reveal specific nucleotide targets of SHM in zebrafish and suggest that AID and affinity maturation contribute to antibody diversification in this emerging immunological model.

Identification and characterization of new long conserved noncoding sequences in vertebrates

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.

Human Genome Ultraconserved Elements Are Ultraselected

Ultraconserved elements in the human genome are defined as stretches of at least 200 base pairs of DNA that match identically with corresponding regions in the mouse and rat genomes. Most ultraconserved elements are noncoding and have been evolutionarily conserved since mammal and bird ancestors diverged over 300 million years ago. The reason for this extreme conservation remains a mystery. It has been speculated that they are mutational cold spots or regions where every site is under weak but still detectable negative selection. However, analysis of the derived allele frequency spectrum shows that these regions are in fact under negative selection that is much stronger than that in protein coding genes.

Purifying Selection Maintains Highly Conserved Noncoding Sequences in Drosophila

The majority of metazoan genomes consist of nonprotein-coding regions, although the functional significance of most noncoding DNA sequences remains unknown. Highly conserved noncoding sequences (CNSs) have proven to be reliable indicators of functionally constrained sequences such as cis-regulatory elements and noncoding RNA genes. However, CNSs may arise from nonselective evolutionary processes such as genomic regions with extremely low mutation rates known as mutation "cold spots." Here we combine comparative genomic data from recently completed insect genome projects with population genetic data in Drosophila melanogaster to test predictions of the mutational cold spot model of CNS evolution in the genus Drosophila. We find that point mutations in intronic and intergenic CNSs exhibit a significant reduction in levels of divergence relative to levels of polymorphism, as well as a significant excess of rare derived alleles, compared with either the nonconserved spacer regions between CNSs or with 4-fold silent sites in coding regions. Controlling for the effects of purifying selection, we find no evidence of positive selection acting on Drosophila CNSs, although we do find evidence for the action of recurrent positive selection in the spacer regions between CNSs. We estimate that approximately 85% of sites in Drosophila CNSs are under constraint with selection coefficients (N(e)s) on the order of 10-100, and thus, the estimated strength and number of sites under purifying selection is greater for Drosophila CNSs relative to those in the human genome. These patterns of nonneutral molecular evolution are incompatible with the mutational cold spot hypothesis to explain the existence of CNSs in Drosophila and, coupled with similar findings in mammals, argue against the general likelihood that CNSs are generated by mutational cold spots in any metazoan genome.

Conserved noncoding sequences are selectively constrained and not mutation cold spots

Noncoding genetic variants are likely to influence human biology and disease, but recognizing functional noncoding variants is difficult. Approximately 3% of noncoding sequence is conserved among distantly related mammals, suggesting that these evolutionarily conserved noncoding regions (CNCs) are selectively constrained and contain functional variation. However, CNCs could also merely represent regions with lower local mutation rates. Here we address this issue and show that CNCs are selectively constrained in humans by analyzing HapMap genotype data. Specifically, new (derived) alleles of SNPs within CNCs are rarer than new alleles in nonconserved regions (P = 3 x 10(-18)), indicating that evolutionary pressure has suppressed CNC-derived allele frequencies. Intronic CNCs and CNCs near genes show greater allele frequency shifts, with magnitudes comparable to those for missense variants. Thus, conserved noncoding variants are more likely to be functional. Allele frequency distributions highlight selectively constrained genomic regions that should be intensively surveyed for functionally important variation.

Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells.

The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. All transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of greater than 99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosine-containing non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.