Missense mRNA

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Missense mRNA is a messenger RNA bearing one or more mutated codons that yield polypeptides with an amino acid sequence different from the wild-type or naturally occurring polypeptide. [1] Missense mRNA molecules are created when template DNA strands or the mRNA strands themselves undergo a missense mutation in which a protein coding sequence is mutated and an altered amino acid sequence is coded for.

Contents

Biogenesis

A missense mRNA arises from a missense mutation, in the event of which a DNA nucleotide base pair in the coding region of a gene is changed such that it results in the substitution of one amino acid for another. [2] The point mutation is nonsynonymous because it alters the RNA codon in the mRNA transcript such that translation results in amino acid change. An amino acid change may not result in appreciable changes in protein structure depending on whether the amino acid change is conservative or non-conservative. This owes to the similar physicochemical properties exhibited by some amino acids. [3]

Missense mRNAs may be detected as a result of two different types of point mutations - spontaneous mutations and induced mutations. [4] Spontaneous mutations occur during the DNA replication process where a non-complementary nucleotide is deposited by the DNA polymerase in the extension phase. The consecutive round of replication would result in a point mutation. If the resulting mRNA codon is one that changes the amino acid, a missense mRNA would be detected. A hypergeometric distribution study involving DNA polymerase β replication errors in the APC gene revealed 282 possible substitutions that could result in missense mutations. When the APC mRNA was analyzed in the mutational spectrum, it showed 3 sites where the frequency of substitutions were high. [5]

Induced mutations caused by mutagens can give rise to missense mutations. [4] Nucleoside analogues such as 2-aminopurine and 5-bromouracil can insert in place of A and T respectively. Ionizing radiation like x-rays and γ-rays can deaminate cytosine to uracil. [6]

Missense mRNAs may be applied synthetically in forward and reverse genetic screens used to interrogate the genome. Site-directed mutagenesis is a technique often employed to create knock-in and knock-out models that express missense mRNAs. For example, in knock-in studies, human orthologs are identified in model organisms to introduce missense mutations, [7] or a human gene with a substitution mutation is integrated into the genome of the model organism. [8] The subsequent loss-of-function or gain-of-function phenotypes are measured to model genetic diseases and discover novel drugs. [9] While homologous recombination has been widely used to generate single-base substitutions, novel technologies that co-inject gRNA and hCas9 mRNA of the CRISPR/Cas9 system, in conjunction with single-strand oligodeoxynucleotide (ssODN) donor sequences have shown efficiency in generating point mutations in the genome. [9] [10] [11]

Evolutionary implications

Non-synonymous RNA editing

Substitutions can occur on the level of both the DNA and RNA. RNA editing-dependent amino acid substitutions can produce missense mRNA's of which occur through hydrolytic deaminase reactions. Two of the most prevalent deaminase reactions occur through the Apolipoprotein B mRNA editing enzyme (APOBEC) and the adenosine deaminase acting on RNA enzyme (ADAR) which are responsible for the conversion of cytidine to uridine (C-to-U), and the deamination of adenosine to inosine (A-to-I), respectively. [12] Such selective substitutions of uridine for cytidine, and inosine for adenosine in RNA editing can produce differential isoforms of missense mRNA transcripts, and confer transcriptome diversity and enhanced protein function in response to selective pressures. [13]

See also

Related Research Articles

<span class="mw-page-title-main">Mutation</span> Alteration in the nucleotide sequence of a genome

In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosis, or meiosis or other types of damage to DNA, which then may undergo error-prone repair, cause an error during other forms of repair, or cause an error during replication. Mutations may also result from substitution,insertion or deletion of segments of DNA due to mobile genetic elements.

Deamination is the removal of an amino group from a molecule. Enzymes that catalyse this reaction are called deaminases.

The coding region of a gene, also known as the coding sequence (CDS), is the portion of a gene's DNA or RNA that codes for a protein. Studying the length, composition, regulation, splicing, structures, and functions of coding regions compared to non-coding regions over different species and time periods can provide a significant amount of important information regarding gene organization and evolution of prokaryotes and eukaryotes. This can further assist in mapping the human genome and developing gene therapy.

<span class="mw-page-title-main">Nucleic acid sequence</span> Succession of nucleotides in a nucleic acid

A nucleic acid sequence is a succession of bases within the nucleotides forming alleles within a DNA or RNA (GACU) molecule. This succession is denoted by a series of a set of five different letters that indicate the order of the nucleotides. By convention, sequences are usually presented from the 5' end to the 3' end. For DNA, with its double helix, there are two possible directions for the notated sequence; of these two, the sense strand is used. Because nucleic acids are normally linear (unbranched) polymers, specifying the sequence is equivalent to defining the covalent structure of the entire molecule. For this reason, the nucleic acid sequence is also termed the primary structure.

<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a branch of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. 

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.

<span class="mw-page-title-main">Point mutation</span> Replacement, insertion, or deletion of a single DNA or RNA nucleotide

A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequences that are moderately predictable based upon the specifics of the mutation. These consequences can range from no effect to deleterious effects, with regard to protein production, composition, and function.

In genetics, a nonsense mutation is a point mutation in a sequence of DNA that results in a nonsense codon, or a premature stop codon in the transcribed mRNA, and leads to a truncated, incomplete, and possibly nonfunctional protein product. Nonsense mutations are not always harmful; the functional effect of a nonsense mutation depends on many aspects, such as the location of the stop codon within the coding DNA. For example, the effect of a nonsense mutation depends on the proximity of the nonsense mutation to the original stop codon, and the degree to which functional subdomains of the protein are affected. As nonsense mutations leads to premature termination of polypeptide chains; they are also called chain termination mutations.

<span class="mw-page-title-main">Germline mutation</span> Inherited genetic variation

A germline mutation, or germinal mutation, is any detectable variation within germ cells. Mutations in these cells are the only mutations that can be passed on to offspring, when either a mutated sperm or oocyte come together to form a zygote. After this fertilization event occurs, germ cells divide rapidly to produce all of the cells in the body, causing this mutation to be present in every somatic and germline cell in the offspring; this is also known as a constitutional mutation. Germline mutation is distinct from somatic mutation.

In genetics, a missense mutation is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution.

<span class="mw-page-title-main">Insertion (genetics)</span> Type of mutation

In genetics, an insertion is the addition of one or more nucleotide base pairs into a DNA sequence. This can often happen in microsatellite regions due to the DNA polymerase slipping. Insertions can be anywhere in size from one base pair incorrectly inserted into a DNA sequence to a section of one chromosome inserted into another. The mechanism of the smallest single base insertion mutations is believed to be through base-pair separation between the template and primer strands followed by non-neighbor base stacking, which can occur locally within the DNA polymerase active site. On a chromosome level, an insertion refers to the insertion of a larger sequence into a chromosome. This can happen due to unequal crossover during meiosis.

<span class="mw-page-title-main">RNA editing</span> Molecular process

RNA editing is a molecular process through which some cells can make discrete changes to specific nucleotide sequences within an RNA molecule after it has been generated by RNA polymerase. It occurs in all living organisms and is one of the most evolutionarily conserved properties of RNAs. RNA editing may include the insertion, deletion, and base substitution of nucleotides within the RNA molecule. RNA editing is relatively rare, with common forms of RNA processing not usually considered as editing. It can affect the activity, localization as well as stability of RNAs, and has been linked with human diseases.

Guide RNA (gRNA) or single guide RNA (sgRNA) is a short sequence of RNA that functions as a guide for the Cas9-endonuclease or other Cas-proteins that cut the double-stranded DNA and thereby can be used for gene editing. In bacteria and archaea, gRNAs are a part of the CRISPR-Cas system that serves as an adaptive immune defense that protects the organism from viruses. Here the short gRNAs serve as detectors of foreign DNA and direct the Cas-enzymes that degrades the foreign nucleic acid.

<span class="mw-page-title-main">ADAR</span> Mammalian protein found in Homo sapiens

The double-stranded RNA-specific adenosine deaminase enzyme family are encoded by the ADAR family genes. ADAR stands for adenosine deaminase acting on RNA. This article focuses on the ADAR proteins; This article details the evolutionary history, structure, function, mechanisms and importance of all proteins within this family.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

<span class="mw-page-title-main">Mutagenesis (molecular biology technique)</span>

In molecular biology, mutagenesis is an important laboratory technique whereby DNA mutations are deliberately engineered to produce libraries of mutant genes, proteins, strains of bacteria, or other genetically modified organisms. The various constituents of a gene, as well as its regulatory elements and its gene products, may be mutated so that the functioning of a genetic locus, process, or product can be examined in detail. The mutation may produce mutant proteins with interesting properties or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of a particular cell function to be investigated.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

<span class="mw-page-title-main">CRISPR gene editing</span> Gene editing method

CRISPR gene editing (CRISPR, pronounced "crisper", refers to "clustered regularly interspaced short palindromic repeats") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.

Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.

Phage-assisted continuous evolution (PACE) is a phage-based technique for the automated directed evolution of proteins. It relies on relating the desired activity of a target protein with the fitness of an infectious bacteriophage which carries the protein's corresponding gene. Proteins with greater desired activity hence confer greater infectivity to their carrier phage. More infectious phage propagate more effectively, selecting for advantageous mutations. Genetic variation is generated using error-prone polymerases on the phage vectors, and over time the protein accumulates beneficial mutations. This technique is notable for performing hundreds of rounds of selection with minimal human intervention.

References

  1. ↑ Jameson JL. Principles of Molecular Medicine. Springer. p. 731.
  2. ↑ Belgrader P, Maquat LE (September 1994). "Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse major urinary protein, while both types of mutations can facilitate exon skipping". Molecular and Cellular Biology. 14 (9): 6326–36. doi:10.1128/mcb.14.9.6326. PMC   359159 . PMID   8065364.
  3. ↑ "Missense Mutation". Genome.gov. Retrieved 2019-11-08.
  4. 1 2 Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000). "Mutations: Types and Causes". Molecular Cell Biology. 4th Edition.
  5. ↑ Muniappan BP, Thilly WG (June 2002). "The DNA polymerase beta replication error spectrum in the adenomatous polyposis coli gene contains human colon tumor mutational hotspots". Cancer Research. 62 (11): 3271–5. PMID   12036944.
  6. ↑ "Mutations | Microbiology". courses.lumenlearning.com. Retrieved 2019-10-09.
  7. ↑ Tessadori F, Roessler HI, Savelberg SM, Chocron S, Kamel SM, Duran KJ, et al. (October 2018). "Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders". Disease Models & Mechanisms. 11 (10): dmm035469. doi:10.1242/dmm.035469. PMC   6215435 . PMID   30355756.
  8. ↑ Robertson NG, Jones SM, Sivakumaran TA, Giersch AB, Jurado SA, Call LM, et al. (November 2008). "A targeted Coch missense mutation: a knock-in mouse model for DFNA9 late-onset hearing loss and vestibular dysfunction". Human Molecular Genetics. 17 (21): 3426–34. doi:10.1093/hmg/ddn236. PMC   2566528 . PMID   18697796.
  9. 1 2 Okamoto S, Amaishi Y, Maki I, Enoki T, Mineno J (March 2019). "Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs". Scientific Reports. 9 (1): 4811. Bibcode:2019NatSR...9.4811O. doi:10.1038/s41598-019-41121-4. PMC   6423289 . PMID   30886178.
  10. ↑ Inui M, Miyado M, Igarashi M, Tamano M, Kubo A, Yamashita S, et al. (June 2014). "Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system". Scientific Reports. 4: 5396. Bibcode:2014NatSR...4.5396I. doi:10.1038/srep05396. PMC   4066261 . PMID   24953798.
  11. ↑ Prykhozhij SV, Fuller C, Steele SL, Veinotte CJ, Razaghi B, Robitaille JM, et al. (September 2018). "Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9". Nucleic Acids Research. 46 (17): e102. doi:10.1093/nar/gky512. PMC   6158492 . PMID   29905858.
  12. ↑ Meier JC, Kankowski S, Krestel H, Hetsch F (2016). "RNA Editing-Systemic Relevance and Clue to Disease Mechanisms?". Frontiers in Molecular Neuroscience. 9: 124. doi: 10.3389/fnmol.2016.00124 . PMC   5120146 . PMID   27932948.
  13. ↑ Yablonovitch AL, Deng P, Jacobson D, Li JB (November 2017). "The evolution and adaptation of A-to-I RNA editing". PLOS Genetics. 13 (11): e1007064. doi: 10.1371/journal.pgen.1007064 . PMC   5705066 . PMID   29182635.
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