Chapter 8 - RNA: Transcription, Processing and Decay

• mRNA transcription and decay mechanisms in eukaryotes are similar to those in bacteria.
• mRNA processing, editing, and modification occur and can affect the abundance and sequence of proteins in eukaryotes.
• siRNAs regulate the abundance of specific RNAs and play a role in maintaining genome integrity in eukaryotes.
• The U1 snRNA recognizes 5' splice sites by base pairing.
• The efficiency of splicing is affected by the strength of U1 snRNA base pairing at the 5' splice site.
• Mutations in the 5' splice site that reduce the number of hydrogen bonds lead to a decrease in the efficiency of splicing.
• Splicing efficiency can be restored by compensatory mutations in the U1 snRNA.
• The U1 snRNA identifies 5' splice sites by base pairing.
• The U1 snRNA binds the 5' splice site and U2 binds the branch point, with the U2 snRNA base pairing to nucleotides across the branch point, except for the key adenosine.
• The splicing reaction begins with stepwise recognition of pre-mRNA sequence elements.
• Spliceosome assembly is completed by entry of the U4, U5, and U6 snRNPs as a preassembled tri-snRNP complex.
• The U1 and U4 snRNPs are released from the spliceosome, the U6 snRNP base pairs to the 5' splice site, and the U5 snRNP base pairs to both exon sequences, placing the splice sites in close proximity.
• Before RNAs are ready to be exported to the cytoplasm for translation, they undergo extensive processing, including deletion of internal nucleotides and addition of special nucleotide structures to the 5′ and 3′ ends.
• Both bacteria and eukaryotes also produce other types of RNA that are not translated into protein but instead perform a variety of roles in cells by base pairing to other RNAs, binding proteins, and performing enzymatic reactions.
• RNAs interact with DNA and other RNAs by base pairing of complementary sequences, and proteins interact with DNA and RNA by binding specific sequences.
• Mutations in DNA and RNA that disrupt molecular interactions can affect the expression of proteins.
• mRNAs are modified with a cap at the 5′ end and a poly(A) tail at the 3′ end, and introns are removed.
• Modifications at the ends of mRNAs increase their stability and assist translation.
• Sequences within a pre-mRNA, along with snRNA or protein factors that bind them, define sequences as intron or exon for splicing as well as dictate the site of cleavage and polyadenylation.
• In bacteria, ribosomes associate with mRNAs as they are being transcribed, whereas in eukaryotes ribosome association and translation can take place only after mRNAs are exported from the nucleus to the cytoplasm.
• The last step in the life cycle of an RNA is decay.
• mRNA decay in both bacteria and eukaryotes occurs via defined pathways that begin with recruitment of specific enzymes.
• In bacteria, an endonuclease is recruited by interacting with a 5′-monophosphate, and in eukaryotes an exonuclease is recruited by interacting with proteins that associate with the 3′ UTR.
• The initiating steps of decay generate recognition sites for further decay by other enzymes.
• In some eukaryotes, very short RNAs such as siRNAs base pair to mRNAs and bring along an endonuclease that initiates decay.
• One of the normal functions of siRNAs is to silence the expression of repetitive genes in genomes such as transposons.
• Researchers have a head start in figuring out how these RNAs are transcribed, processed, transported, and decayed as well as how they function, because it is likely that they have similar life cycle stages.
• Co-suppression phenotypes result from the insertion of a transgene that controls pigmentation into the genome of a wild-type petunia.
• Fire and Mello injected C. elegans with RNAs that were identical in sequence to an endogenous gene that when mutated causes adult worms to twitch.
• Injection of dsRNA caused a much stronger twitcher phenotype, demonstrating that dsRNA mediates suppression of endogenous gene expression in a process that is now called RNA interference (RNAi).
• RNAi is very specific, and only RNAs with perfect complementarity to the dsRNA are affected.
• RNAi is extremely potent, as only a few dsRNA molecules are required per cell to inhibit expression of the targeted gene, indicating that the process is catalytic.
• RNAi can affect cells and tissues that are far removed from the site of introduction, indicating that there is an RNA transport mechanism.
• RNAi affects the progeny of injected animals, indicating that the targeting information is heritable.
• RNAi has had a tremendous impact on almost all fields of biology research through its use as a tool to perform loss-of-function experiments.
• RNAi silences gene expression by targeting RNAs for decay in the cytoplasm of cells.
• Capping of mRNAs is programmed to occur early in transcription through the association of capping enzymes with phosphorylated serine 5 on the CTD of RNA polymerase II.
• Polyadenylation of mRNAs involves the sequential action of two enzymes: cleavage, which cuts the mRNA away from the transcribing RNA polymerase II, and polyadenylation, which adds 50–250 adenosine (A) residues to the end of the cleaved mRNA.
• Sequence elements within the 3′ UTR determine where cleavage occurs in humans, the highly conserved six-nucleotide (hexanucleotide) sequence AAUAAA is located 10–30 nucleotides upstream of the cleavage site, also known as the poly(A) site.
• The AAUAAA is important for both cleavage and polyadenylation because it is bound by a protein complex called Cleavage and Polyadenylation Specificity Factor (CPSF), which contains the endonuclease enzyme that executes the cleavage step.