Non-coding/regulatory RNA Flashcards
MicroRNA discovery (profiling methods):
1) qRT-PCR 2) mRNA microarray 3) Nanostring 4) RNA-seq
_________ of miRNAs frequent event in cancers
Downregulation
52.5% located in areas of genome associated with development of cancer called _________
Cancer associated genomic regions (CAGR)
SiRNAs as therapeutic targets challenges:
RNA half-life, delivery system, immune response and toxic effects, delivery to tumor, uptake by tumors, specific target recognition
Primary structure RNA:
Sequence of nucleotides from 5’ end to 3’ end
Secondary structure RNA:
Hydrogen bonds formed within molecule
Tertiary structure RNA:
Locations of the atoms in 3D space
RNAi
RNA interference
RNA interference:
Silencing of gene expression by different types small RNAs (18-30 nt) piRNAs, siRNAs, miRNAs
RNAi results in (4):
Translational inhibition, mRNA degradation, transcriptional silencing, chromatin remodeling
small RNAs common features:
20-30 nt, associated with Argonatue (AGO) fam proteins
AGO protein family subclades:
AGO, PIWI, WAGO
PIWI subclade:
Associated with piRNAs, germ-cell specific
AGO subclade:
miRNA, siRNA, piRNA, ubiquitously expressed
Main biological role piRNAs:
Restrain spread of selfish genetic elements in germ cells, ex. transposons
piRNA clusters correspond to _________
Transposon-rich regions
piRNAs arise from __________
ssRNA precursors, independent of RNase III enzyme
piRNAs biogenesis:
Primary piRNA processing pathway and amplification loop (Ping-pong cycle)
siRNAs vs miRNAs
Both 18-25 nt, siRNA silencing target deg, miRNA silencing translational inhibition or deg
siRNAs induce _________ and ________
mRNA cleavage (Dicer, RISC-AGO) and chromatin remodeling (RITS, Chp1)
miRNA characteristics:
Processed from long endogenous ssRNA, RISC containing AGO protein, guides binding of RISC to 3’UTR of target mRNA leading to post-transcrip silencing
miRNA location:
Intronic majority, more than 50% transcribed polycistronic, primary transcript molecules (pri-miRNAs) fold into imperfect dsRNA-like hairpins
pri-miRNA structure
Basal segments->lower stem(11 bp)->cleavage by Drosha->upper stem (22 bp)->cleavage by Dicer->terminal loop
Drosha (pri-miRNA Step 1)
RNAse III endonuclease found in nucleus, functions in combo with DGCR8, generates 65 bp product with 3’ overhang
DGCR8
Double stranded RNA binding protein
Exportin and Ran-GTP role (pri-miRNA Step 2)
Export pri-miRNAs to cytoplasm
Dicer (pri-miRNA Step 3)
RNase III-like endonuclease, works in complex with TRBP, final 22 bp miRNA duplex with 3’ overhang
TRBP
dsRNA binding protein
let-7 role
Regulates Dicer expression in negative feedback loop
RISC (pri-mRNA Step 4)
miRNA duplex incorporated into microRNA ribonucleoprotein complex, mature miRNAs one strand selected for function
Main function of AGO subclade:
Expose and present miRNAs to target RNA
Non-canonical pathway miRNA
Mirtron (short introns that after transcription are made into miRNA mimics)
miRNA-mRNA interactions in animals
miRNA pair imperfectly with mRNA targets, usually in 3’UTR, induce translational repression
miRNA-mRNA interactions in plants
miRNAs base pair near perfect complementarity to mRNA targets, trigger endonucleolytic cleavage of mRNA similar to siRNAs
miRNA-mRNA silencing mechanisms (post-transcriptional)
1) Translational repression (5’ cap binding or elongation block) 2) mRNA turnover (shortening of polyA tail or removal of 5’ cap and deg) 3) endonucleolytic cleavage
miRNAs in vertebrates
Abundant and highly conserved, repress gene expression, tissue specific expression (zebrafish), cell proliferation and differentiation
IsomiRs/Sequence heterogeneity
miRNA sequences that have variations with respect to the reference sequence
