OBJ - RNA Metabolism Flashcards
Understand the basic mechanism of pre-mRNA splicing, including the sequences and information that define the splice sites.
- nascent pre-messenger RNA pre-mRNA
- removal of introns & joining of exons
- catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs)
Mechanism = Double Transesterification
- Hydroxyl on intron attacks nucleophilic P on exon 1 on the 3’ C
- Exon 1’s 3’ end OH attacks P on exon 2
- Exon1-P-Exon 2 & Lariat intron
Define alternative splicing and understand how it increases protein diversity.
- Alternative Splicing (~90% of genes)
- Results in a single gene coding for multiple proteins
- Increases diversity & variability for 1 genetic code
Ex: Allows 1 cell to secrete & have membrane bound forms of protein with the same variable region of RNA or 2 different peptides
Understand the different mechanisms by which mutations can give rise to genetic diseases.
15-50% Splicing Defects
Mutations in splicing enhancers/silencers cause disease not point mutations (often silent)
Cause wrong splice sites for introns/exons; may added or subtracted
Know how the example disease, Spinal Muscular Atrophy, occurs and how understanding of its molecular biology is likely to lead to effective treatments
Spinal Muscular Atrophy (SMA) is a genetic, neuromuscular disease characterized by dysfunction and death of motor neurons in the spinal cord, generally leading to death in childhood.
The incidence of SMA is 1 in 10,000 (among the most common genetic causes of infant mortality.)
There is no current treatment. (only palliative care)
SMA is caused by a mutation of the SMN1 gene (SMN = Survival of Motor Neurons).
Both copies of SMN1 must be mutated to generate the disease; SMN 2 cannot substitute for SMN 1
Changes exonic splicing enhancer -> SR protein no longer binds, resulting insufficient recruitment & skips exon 7 -> SMN delta7 (nonfunction, unstable)
Mutation is a silent mutation -> C to T, but still Phe SNM2 not spliced properly
Treatment: try to block hnRNP by blocking ISS with an oligonucleotid; allows SR protein to recruit U1 & U2 (splicing factors) -> corrects successfully in lab & with mice
**SMN required for assembly of snRNPs (U1-U6)
-still unknown why defect in SMN causes SMA & is seen in nerve cells
4 total alleles = 2 SMN 1’s & 2 SMN 2’s
- SMN1 = dominant; so need both to be nonfunction so SMN2’s defect is expressed/manifested
- SMN2 = defective copy of SMN 1
Spilcing Defects
Large target size with intronic/Exonic splicing enhancers/silencers
Spliceosome
snRNPs = small nuclear Ribonucleoprotein particles
- U1-U6
U2 & U6 = catalytically active protion
followed by 2 transesterifications -> spliced exons & lariat intron
(Exon 1/upstream exon = AG)/GUAAGU
NCAG/(G = Exon 2/downstream exon )
Branch Site (intron)
they contain a branch point, a particular nucleotide sequence near the 3’ end of the intron that becomes covalently linked to the 5’ end of the intron during the splicing process, generating a branched (lariat) intron
Splicing Enhancers/silencers
Exonic/Intronic Splicing Enhancers:
-binding proteins that recruits U1 & U2 to splice the sites
Exonicx/Intronic Splicing Silencers
- binding proteins that interfere with the ID of the splice site
SR protein
- RECRUITS SPLICING FACTORS
- RNA binding domain + RS domain (activation domain)
- bound to ESE/ISE; recruits the splicing machinery -> U1 & U2 snRNPs to initiate splicing
- each has an RNA binding domain (RRM) that binds to specific RNA sequences & RS domain
hnRNP proteins
- bind to silencers (ISS & ESS)
- blocks SR protein recruiting splicing factors