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RNA Editing

-process by which mRNA transcript is altered by cellular enzymes prior to its export to the cytoplasm where it will be translated

-during editing process four different types of changes to mRNA an occur

-(1) deamination of C to U

-(2) deamination of A to I

-(3) insertion of nucleotides

-(4) deletion of nucleotides -these edits change sequence of bases that are found in mRNA transcript

-during translation these can lead to changes in amino acid sequence of proteins as well as removal or addition of stop codons


RNA Editing Discovery

-discovered when researchers identified mRNAs that had little to no resemblance to any sequence within genome

-subsequently identified enzymes that mediate the four types of editing listed before

-however, DNA bases can also be deaminated as a result of existing in an aqueous environment or through mistakes made by deaminase enzymes

-C-U and A-I deaminations within DNA are removed via base excision repair machinery

-such base conversions are not reversed if they are found within mRNA transcript

-also seen that replication slippage can cause bases to be added or deleted from the DNA template strands


Editing Cytosine Residues to Uracil

-one of most famous examples of RNA editing inC-U deamination of human apoliproprotein B (apoB) mRNA transcript

-apoB gene is transcribed in both human liver and intestinal tissue

-within liver transcript is unedited

-within in intestinal cells a single cytosine residue is deaminated to yield a uracil residue -changes the CAA codon to UAA

-CAA codes for amino acid glutamine while UAA is a stop codon

-so within intestinal cells a shorter protein is produced while the unedited transcript will code for larger 4563 aa protein within cells of liver

-these proteins have very different functions thus RNA editing serves as another avenue to generate protein diversity without having to encode additional genes into genome


apoB Specificity (Mooring Sequences and 3' Efficiency Sites)

-transcript is over 14.000 nucleotides long yet only single cytosine residue is edited

-sequences within mRNA transcript called "mooring sequences" and "3' efficiency elements" are thought to be important for ensuring that only certain nucleotides are edited

-efficiency element consists of nucleotides that are complementary to the region that needs to be edited

-allows for the formation of localized regions of double-stranded RNA which appears to be a prerequisite for editing

-mooring sequence (10-11 nucleotides in length) is usually located 4-6 bases downstream of the base to be edited

-serves to recruit the enzymes that will actually carry out nucleotide editing -in humans this enzyme complex is called APOBEC-1 -5` efficiency element has also been identified but its role remains somewhat unclear.


Editing Adenosine Residues to Inosine

-mechanism involves the deamination of adenosine bases to a special nucleotide called inosine

-this nucleotide found within tRNAs (most commonly within the anti-codon portion of the tRNA molecule)

-allows for greater flexibility in base-pairing and can also change the amino acid composition of proteins

-normally adenine pairs with uracil

-once adenine nucleotide as been deaminated to inosine the inosine nucleotide will now base pair with cytosine

-inosine nucleotides can also base pair with uracil and adenine nucleotides as well.

-can lead to dramatic differences in which tRNAs are recruited to the mRNA transcript


A-I Deamination Reaction

-also requires the formation of a double stranded RNA molecule

-this case, the complementary sequence is found within intronic sequences of the mRNA

-so A-I editing must occur prior to RNA splicing while C-U editing can occur before, during and after splicing

-formation of double-stranded RNA near the base that is to be edited serves as a signal for ensuring specificity of the A-I deamination process


Editing Adenosine Residues to Inosine (Background)

-much more common than C-U editing

-examples in which up to 50% of all adenine nucleotides are converted to inosine

-also examples in which only a single adenine nucleotide is edited. A

-precision of A-I editing is in part determined by the presence of sequences within introns that can form complementary base-pairs with exon sequences


Editing of Adenosine Residues to Inosine (Process)

-carried out by set of enzymes called adenosine deaminases acting on RNA (ADARs)

-these enzymes will recognize and bind ot double-stranded RNA irrespective of its sequence content

-ADAR proteins found in nearly all eukaryotes (flies and humans)


Most Famous Examples of Transcripts Subject to A-I Editing

-include mRNAs that code for voltage-gated ion channels and neurotransmitter receptors.

-ex: human GluR2 receptor is an ion channel that can be opened in the presence of glutamine

-opening of the channel allows for calcium, sodium and potassium to enter the cell thus altering the voltage across the lipid bilayer

-channel plays important roles in neuronal physiology

-GluR2 mRNA transcript undergoes A-I editing at a single site

-DNA sequence is predicted to encode a Gln amino acid

-RNA editing changes it to Arg. Other receptors that are edited include the serotonin receptors

-mRNA transcripts are edited in several locations which then results in changes to several amino acids within one of the cytoplasmic loops of the receptor


Editing by Insertion of Nucleotides

-also alters mRNA transcripts

-insertion of nucleotides will change the amino acid content of the translated protein


Insertion of Uracil Residues

-mediated by guide RNAs (gRNAs), which are non-coding RNAs (ncRNA) that are encoded by separate genes within the genome

-gRNAs are usually short in length (<100 nucleotides) and can form double-stranded RNA with mRNA transcripts through hydrogen bonding between complementary sequences

-each gRNA can be divided into three regions: the anchor region, the editing region and a poly U tail

-guide RNA will contain several adenine nucleotides that will not base pair correctly with the mRNA transcript

-these are pushed out to form a short bubble

-cell recognizes the bubble and attempts to fix the mRNA transcript

-first step is for an endonuclease to make a single strand nick on the mRNA.

-followed by the incorporation of uracil nucleotides (by a special RNA polymerase) into the mRNA transcript at positions that are complementary to the adenine nucleotides within the gRNA.

-RNA specific ligase is able to then seal the mRNA transcript by forming phosphodiester bonds.

-uracil nucleotides can also be deleted by using similar mechanisms

-in these cases the gRNAs will have fewer nucleotides and this will force the cell to remove nucleotides from the mRNA transcript


Nuclear Export

-eukaryote mRNA transcripts are exported to the cytoplasm after they are spliced and edited

-nuclear exportation of mRNAs is a regulated process

-once transcript is ready for export it will be bound by a set of RNA binding proteins

-main residence for these factors is the nucleus but they are able to temporarily move into the cytoplasm to deposit their cargo (the mRNA transcript).

-once they dump the mRNAs in the cytoplasm they are immediately moved back into the nucleus


RNA Binding Proteins in Nuclear Export, NES and NLS

-able to shuttle back and forth between the nucleus and the cytoplasm because the contain sets of amino acids that are called Nuclear Export Sequences (NES) and Nuclear Localization Sequences (NLS).

-while RNA binding proteins need both sequences certain proteins such as DNA binding proteins will only have NLS sequences.

-Green Fluorescent Protein being localized to the nucleus (picture).

-was fused to a strong NLS sequence that was taken from a DNA binding protein

-if an NLS sequence is mutated then a transcription factor will not be localized to the nucleus.

-if an NES sequence is mutated then this could affect the ability of an RNA binding protein from transporting an mRNA transcript to the cytoplasm.


Nuclear Pores

-both RNAs and proteins exit the nucleus through openings these

-comprised of dozens of proteins that function as gates to prevent inappropriate entrance into or exit from the nucleus

-nuclear import and exportation proteins will physically interact with proteins of the nuclear pore

-biochemical interaction is critical for the opening and closing of the pore