Noncoding RNA Flashcards
Classes on non coding RNAs
tRNAs rRNAs Spliceosomal RNAs snoRNAs piRNAs Endogenous siRNAs Enhancer RNAs miRNAs lincRNAs
DNA –>RNA–>Protein
DNA-->DNA = DNA replication DNA-->RNA = transcription (snRNAS) RNA-->DNA = reverse transcription RNA-->RNA = RNA replication RNA-->protein = translation Protein--> protein = prion propagation
Transfer RNAs
Responsible for translating codon–anticodon information into specific amino acids
How many tRNAs in human genome?
About 500
What was the first discovered noncoding RNA?
Yeast alanine tRNA(1965)
Cloverleaf Crystal structure first determined by X-ray crystallography in 1975
Ribosomal RNAs
Along with ribosomal proteins, makes up the molecular machine that carried out translation
2 RNAs make up more than half the mass of the eukaryotic ribosome
Large subunit catalyzes peptide bond formation
Role of RNAs in splicing
Spliceosomal
Self splicing introns
Spliceosomal RNA
Spliceosome made up of 5 smaller nuclear ribonuclear proteins (snRNPs =snRNA + protein)
RNAs are involved in sequence recognition and possibly catalysis
Self splicing introns
The intron itself forms ribozyme that catalyzes splicing (Cech 89)
Heterochronic mutants
Mutations that disrupted developmental timing in C.elegans
Two mutants : Lin-14 and Lin-4
lin-4 and lin-14
Acts as negative regulator of Lin-14
Ambros: Lin-14 alleles are completely epistatic to lin-4 alleles.
Turns out
Lin-4 represses lin-14 by binding to multiple complimentary strands of 3’UTR of lin-14
MicroRNAs
> 1000 miRNAs have been discovered in the human genome
Lin-4
Can be transcribed as pre-miRNA or derived from introns of pre-mRNAs
Processed by Drosha or Spliceosome to form characteristic hairpin structure
Exported to the cytoplasm
Pre-miRNAs are cleaved by Dicer–>~22nt dsRNA
miRNA is incorporated into RNA-induced silencing complex (RISC) —gene silencing
Gene silencing
Can occur through translation inhibition or target mRNA degradation
miRNA discovery
Shotgun sequencing of small RNA molecules
Stem loop prediction
Covariance of substitution patterns
Ambros
Predicted stem loops from genome sequence sequence and cloned 15 miRNAS
Tuschl
Sequenced small RNAs form worms, flies, and humans
Bartel
Sequenced 330 small RNAs to identify 55 new miRNAs
miRNA target prediction
Computational predictions
1) Strong complementary between mRNA 3’UTR and miRNA (over seed region)
2) evolutionary constraint of seed region
3) accessibly secondary structure at UTR target sites
4) expression profiles of mRNAs and miRNAs
miRNA target prediction
Experimental predictions
Over expression and knock down experiments
HITS-CLIP and related techniques
NGS library construction tricks to clone mRNA degradation products
Long non-coding RNAs
> 200 bp
Transcribed by PolII, have poly A tails
Often lowly expressed, cell type restricted
Often poorly conserved
Tiling microarrays and ngs
Allowed interrogation of genome wide transcription patterns
Discovery lncRNAs
Expression: tiling microarrays and NGS Chromatin modification signatures -H3K4me3 at promoters -H3K36me3 through gene body Evolutionary conservation Differential expression
Does lncRNAs contain an open reading frame?
Difficult to interpret because short open reading frames are likely to frequently occur by chance
Are lncRNAs translated ?
Difficult to asses but modern. Proteomics approaches may begin to shed some light
Ribosomal profiling experiments gave been difficult to interpret
Pattern of evolutionary conservation takes advantage of differences between synonymous and no summits substations
XIST
LncRNA
Mediates X chromosome silencing in mammals
Recruits PRC2 to form large repressed H3k27me3 domains across most of the X
HOTAIR
Is expressed from the HoxC cluster, represses HOxD transcription in trans by enhancing recruitment of PRC2
H19
Imprinted gene, involved in cell proliferation and body weight control
Mechanism unknown
NRON
Regulates nuclear export
How do lncRNAs function?
Functional roles maybe as diverse as proteins
Evidence from eQTL studies suggest that they do not often regulate genes in trans
