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RNA editing
post or co-transcriptional alteration of the sequence of nucleotides in the RNA
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define: epitranscriptome
- the collective term for RNA modifications.
- The genome level studies of RNA modifications are known as epitranscripomics
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What are some methods of RNA modification detection?
- Immunoprecipitation with modification-specific antibodies (RIP-seq)
- Chemical treatments (e.g., with click chemistry) that block reverse transcription (Chem-seq)
- Next-gen sequencing: Illumina to locate RT errors in cDNA or Oxford Nanopore to detect electric signal deviations in RNA squiggles
- Chemical derivatization is used: purified RNA is treated with carbodiimide CMCT (B) that reacts with pseudouridine (A) and forms a bulky adduct (C)
- The adduct can be detected by mass-spec or by reverse transcription and sequencing (D)
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Four potential consequences of RNA editing:
- A nucleotide substitution in the 5’UTR region of mRNA
- Editing an intron
- A nucleotide substitution in the coding region
- Editing of a residue in the 3’UTR
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What is a potential consequence of the RNA editing: nucleotide substitution in the 5'UTR region:
- alter its structure and thereby affect translation efficacy
- introduce a novel in-frame start codon (AUA→AUG): extend N-terminus
- remove the original start codon (AUG→GUG): shorten N-terminus
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What is a potential consequence of the RNA editing: nucleotide substitution in the 3'UTR region:
- alter mRNA stability, which depends on AU-rich elements
- affect mRNA trafficking by mutating the ‘ZIP-code’
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What is a potential consequence of RNA editing: editing in an intron:
- introduce a new splice donor site, converting part of the intron to an exon
- alter the structure of the intron, which may affect splice site
- election
- remove existing splice donor/acceptor site: eliminate exons
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What is a potential consequence of RNA editing: nucleotide substitution in the coding region:
- result in a point mutation (amino acid substitution)
- introduce a premature stop-codon, truncating the protein
- remove existing stop codon, extending the protein
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How is RNA editing by insertion/deletion mediated?
- by guide RNA (gRNA) molecules that base-pair with the RNA to be edited and serve as a template for the addition (or removal) of nucleotides in the target:
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How is RNA editing (by Insertion/Deletion) initiated?
- by endonucleolytic cleavage of the pre-mRNA at a site programmed by gRNA-pre-mRNA mismatch that forms a bulge upon basepairing:
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Describe the process of insertion/deletion via RNA editing?
- Deletion reaction (left): U residues are removed by an exonuclease from the 3' end of the 5' cleavage product
- Addition reaction (right): U residues from a UTP donor are added to the 3' end of the 5' cleavage product by a terminal uridylyl transferase (TUTase)
- RNA ligase joins modified fragments
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What direction does Insertion/Deletion editing of RNA occur in?
3' -> 5' direction
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Why did RNA modification/editing evolve?
- To compensate for frame-shift mutations (e.g., CoxII cytochrome oxidase indel editing)
- To correct for suboptimal base-pairing, folding, protein binding (e.g., rRNA pseudouridylation)
- To diversify the function of the gene (e.g, ApoB)
- To regulate translation, splicing, targeting, stability, etc. of mRNA (e.g., m6A)
- As an innate immunity mechanism against viruses (e.g., A-to-I editing
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What percentage of the genome is transcribed?
up to 90% of a eukaryotic genome is transcribed on one or both strands
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What is ncNRA?
- Non-coding RNA (ncRNA) are RNA molecules that function without being translated into a protein
- The most prominent examples of non-coding RNAs are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation
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examples of small ncRNA (<200nt)
- functional housekeeping RNA (rRNA, tRNA, snRNA)
- regulatory (miRNA, siRNA, piRNA, CrasiRNA, telsRNA)
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examples of long ncRNA (>200nt)
- Structure (lincRNA, ecricRNA, TUCRNA, eRNA, NAT, ciRNA, ElciRNA)
- Action (CisRNA, CeRNA, TransRNA)
- Location (intergenic, Bidirectional, Sense, Intronic, Antisense)
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What are the three main lines of thinking to explain why RNA polymerase sometimes initiates and elongates non-coding transcripts instead of transcribing the proper protein coding gene (and which is the most likely reason?)
- (I) Majority of ncRNAs produced by pervasive transcription are non-functional and just transcriptional noise or spurious transcription generated by the RNA polymerase
- (II) Some but not all of the non-coding transcripts have specific functions
- (III) All ncRNAs have functions that are related to the organism’s complexity
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lncRNA
long non-coding RNA
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How long are lcRNAs and what are some known to interact with?
- >200 nt long
- DNA, RNA, and/or protiens
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Describe some known functions of lncRNA
- Signaling: The transcription of certain lncRNAs is very tissue and temporal specific. Their expression can be in response to certain stimuli, such as cellular stress and temperature. Thus, lncRNAs can serve as molecular signals and can act as markers of functionally significant biological events.
- Decoys: The molecular decoy type of activity takes place when specific lncRNAs are transcribed and then bind to and titrate away protein factors. Decoy lncRNAs can "sponge" protein factors such as transcription factors and chromatin modifiers. This leads to broad changes in the cell's transcriptome.
- Guides: LncRNAs can be molecular guides by localizing particular ribonucleoprotein complexes to specific chromatin targets. This activity can cause changes in gene expression either in cis (on neighboring genes) or in trans (distantly located genes) that cannot be easily predicted by just the lncRNA sequence itself.
- Scaffolds: Assembly of complex protein complexes can be supported by lncRNAs, linking factors to together to form new functions. Some lncRNAs possesses different domains that bind distinct protein factors that altogether, may impact transcriptional activation or repression.
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What are the four archetypes of lncRNA mechanism of action:
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circRNA
- circular RNA
- formed from pre-mRNAs via backsplicing of introns/exons
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What is RNAi?
- RNA-dependent gene silencing process
- It is a biological response to double-stranded RNAs (dsRNA) that results in the degradation of homologous mRNA, inhibition of translation, or chromatin silencing
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When does RNAi occur?
- when cells encounter dsRNA from:
- Virus
- Transposon or transgene
- Artificial injection, transfection, particle bombardment, etc.
- Natural co-expression of sense and antisense transcripts
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- Basic mechanism of RNAi
- Long dsRNA molecules are cleaved to produce siRNA by endonuclease Dicer
- siRNA molecules are then incorporated into a multiprotein RNA-induced silencing complex (RISC), that consists if Ago and other proteins
- The anti-sense strand guides RISC to the complementary mRNA for subsequent endonucleolytic cleavage (or translation inhibition)
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