What are microRNAs?
family of small RNAs -> 21-25 nt
prevent expression of genes by binding to mRNAs
What was the first miRNA?
lin-4 -> 22nt ncRNA
bind to 3’UTR of lin-14 mRNA -> repressed lin-14 translation
Explain the miRNA biogenesis.
RISC -> RNA-Induced Silencing Complex
What are the two different methods that miRNAs can inhibit gene expression?
prevent translation
mRNA cleavage
Where is miRNA DNA found in the genome?
solo miRNA -> own promoter
clustered miRNAs -> multiple
intronic miRNAs
mirtrons -> dont need Drosha -> use Splicing machinery
3’ tailed mirtrons
Explain a computational method for predicting miRNA genes in C.Elegans.
miRscan
sliding window approach (110nt)
discard sequences:
known coding
repetitive elements
wrong base composition
fold sequences to secondary structure (RNAfold)
check for stem-loop miRNA structure
How many miRNAs are in the human genome?
2300 mature miRNAs (2019)
Which algorithm is used to predict mammalian miRNA targets? Describe the general workflow.
TargetScan
thermodynamics-based RNA:RNA duplex interactions
comparative sequence analysis
Input:
miRNA that is conserved in multiple species
set of 3’UTR sequences from these species
Method:
check miRNA seed region (2-8 bases) perfect complementarity to 3’UTR
extend to G:U pairs but no mismatches
assign folding free energy G to miRNA:target
assign Z score to each UTR
sort UTRs by Z score -> assign rank
compare organisms -> conserved miRNAs
What are some limitations of TargetScan?
targets have mismatch in seed
could be outside of 3’UTR (plants)
may not be conserved in all organisms
—> actual number of targets is probably much higher
Name three differences between plant and animal miRNAs.
Feature
Plants
Animals
number
100 - 200
100 - >500
target-site
ORF
3’UTR
binding sites in target
one
multiple
location
intergenic
intergenic, intronic
miRNA cluster
uncommon
common
biosynthesis
Dicer-like
Drosha, Dicer
repression
cleavage
repression, cleavage
inhibited genes
development, enzymes
development, enzymes, structural proteins
Which protein complex is associated with the transformation of pre-miRNA in animals?
DICER
Name a database that stores miRNAs.
MiRBase
sequences
targets
nomenclature
Explain the terms multiplicity and cooperativity in the context of miRNAs.
multiplicity: one miRNA can target more than one gene
cooperativity: one gene can be controlled by more than one miRNA
Name a different tool for miRNA-target prediction and briefly describe the approach.
miRanda: Uses sequence complementarity and free energy, does not prioritize conservation.
TargetScan: Focuses on seed region complementarity and evolutionary conservation.
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