601 Translational regulation

5’ capping

7-methylguanosine added to 1st nucleotide

mRNA stability, transport to cytoplasm, protein translation

3’ polyadenylation

mRNA stability, transport to cytoplasm, protein translation

AAUAA polyadenylation signal triggeres mRNA cleavage, poly-A tail added

mRNA decay

enzymatic complex identifies PolyA tail and begins degradation

separate complex decaps the 5’ end

RNA is degraded in the P-body

RNA binding proteins

regulate mRNA stability

alternative promoters

tissue specific isoforms present from a single gene with a variety of

exon inclusion

a gene can have multiple start sites, each one includes different exons therefore different sequences and different proteins

dystrophin

DMD, xlr

79 exons

at least 7 different promoters can be used resulting in tissue specific isoforms

tissue specific enhancers

act on different promoters likely due to biochemical compatibility

pre-mRNA splicing

process in which intronic RNA segments are snipped out and the exonic RNA segments are spliced together

spliceosome

A protein-RNA complex that performs splicing by assembling around the intron and catalyzing reactions to cleave pre-mRNA.

spliceosome is made of

5 snRNPs (U1, U2, U4, U5, U6) and over 60 other proteins

snRNPs

small nuclear ribonuceloproteins

U1 snRNP

bind to 5’ splicing site

U2 snRNP

bind to the branch point

U2AF

bind to the polypyrimidine tract and 3’ splice site

tri-snRNPs

U4 U5 U6

Alternative splicing

results in the production of multiple protein isoforms form a single gene locus

splicing activator/repressor

regulates alternative splicing

intron retention

type of alternative splicing
An intron is retained in the mRNA, which can lead to various outcomes such as nonsense-mediated decay or alternative protein isoforms.

Splicing activator

causes the exon inclusion by promoting the binding of U1 to 5’ and U2AF to 3’

splicing repressor

causes the exon skipping by inhibiting the binding of U1 to 5’ and U2AF to 3’

genetic disease caused by RNA mis-splicing of Lamin A/C (LMNA)

Limb girdle muscular dystrophy

Hutchingson-Gilford progeria syndrome

Dilated cardiomyopathy

INK4a/ARF

example of alternative promoter and alternative splicing

Antisense oligonucleotide

Single-stranded DNA or RNA snippets that alter RNA splicing or trigger degradation of target mRNA.

Antisense oligonucleotide as potential therapies

can mask the binding of proteins to restore normal function

steps of translation

initiation (rate limiting)

elongation (dehydration reaction)

termination (stop codon)

eIF4F complex

regulates the translation initiation by recruiting the 40s subunit, the recruiting 60s forming complete 80s ribosome

True

t/f codon code varies per organsim

most variable codon postion

3rd postion
causes most genetic diversity between people

Mammalian target of rapamycin (mTOR)

key regulator of protein translation
promotes global protein translation

downregulation can stop translation

miRNA

~22 nt long

most abundant class of small non-coding RNA, >2500 in the human genome

regulate >60% of human protein coding genes

bind in the 3’ UTR, coding region, 5’ UTR of target genes

miRNA biogenesis

primary transcript pri-miRNA

Drosha/DGCR8 protein complex cleaves a pri-miRNA to pre-mRNA

Dicer cleaves the hairpin loop of pre-miRNA yielding a miRNA duplex

one strand of the duplex is loaded into the RNA-induced silencing complex (RISC) and guides the RISC complex to target mRNA

repression of translation initiator

interfere with elF4E binding to the 5’ cap structure of mRNAs

inhibition of 80s complex assembly by blocking 60s subunit joining

degradation of target mRNA

complete match between miRNA and target mRNA will cause

long noncoding RNA (LncRNA)

noncoding RNA over 200 nts long

plays a role in controlling gene expression

mechanisms of LncRNAs

act as a sponge to regulate miRNAs

recruit transcription factors

recruit chromatin modifiers

regulate chromatin looping

regulate RNA splicing

siRNAs

~21 nts long

dsRNA

highly specific for gene of interest

siRNA silencing

one strand is loaded into RNA induced silencing complex (RISC) and guides RISC to target mRNA

RISC complex mediate site specific cleavage/destruction of target mRNA

Patisiran

first siRNA-based drug

used to treat peripheral nerve disease (polyneuropathy) incited by hereditary transthyretin-mediated amylodisis (hATTR)

siRNA targeting the mRNA of mutated transthyretin protein

delivery- the siRNA is encapsulated in a lipid nanoparticle

approved by FDA in august 2018

Givosiran

second siRNA based drug

used for the treatment of acute hepatic porphyria (AHP), a genetic disorder resulting in the buildup of toxic porphyrin molecules formed during the production of heme

siRNA targeting amniolevuliante synthase 1, an enzyme critical in heme production

CRISPR Cas-9 for gene editing

can design oligos to recognize the enzyme

produced ds breaks to remove mutation

actually editing the genome

RNA editing

the changes that take place in the RNA sequence after transcription

C to US mediated by deaminase ApoBECs
A to I (inosine) mediated by deaminase ADARs

can produce a trucated product