W9L2 eRNA, circRNA Flashcards
Problem with standard RNA-sequencing
Identifies steady state RNA present in a cell/tissue
* Will not identify transcripts lacking poly(A) tail if using poly(A) enrichment
* May not identify rare RNA or those with short half life
* Will not identify RNA with modifications that interfere with reverse transcription or adaptor ligation
Problem with measuring RNA transcript
Steady state levels of RNA do not accurately mirror transcriptional activity
RNA abundance determined by numerous factors – processing efficiency, RNA stability, miRNA activity
Nuclear run-on experiments map sites in the genome that are transcriptionally engaged
Nuclear run on assay
- Isolate nuclei and pause transcription (ice incubation)
- Introduce a nucleic acid label( Br-UTP run on)
- Restart transcription
- Isolate nascent transcripts and sequence using immunoprecepitation
Global run-on sequencing (Gro-seq) results
Find that transcription occurs upstream of the annotated TSS
Some genes have a peak of transcription at the Transcription Start Site
RNA pol II pausing
Peak of seq reads seen at some TSS is due to pausing of RNA pol II
-30% of human gene are paused. Much lower transcript than initial binding to the TSS
-New way of gene regulation: pausing of RNA elongation by regulatory protein (NELF)
Small RNAs arise from enhancer
Study of mouse neuronal enhancers revealed 1000s bound by RNA pol II and bi-directionally transcribed
Mapping TSS of expressed regions of the human genome
-Study of human cell types and tissues revealed 43K enhancers bound by RNA pol II and bi-directionally transcribed
Characteristic of eRNAs
2D-eRNAs (200bp-2kb)
§ Bidirectional transcription at a high rate
§ Short-lived (half-life = minutes)
§ Capped, non-polyadenylated, non-spliced
§ Function in cis
1D-eRNAs (enhancer-associated lncRNAs) (4-5kb)
§ Unidirectional transcription
§ Longer-lived
§ Capped, polyadenylated, spliced
§ Can function in cis or trans
Possible roles for eRNAs
There are two possible roles for eRNAs
1. eRNA synthesis facilitates delivery of RNA pol II to corresponding promoter by tracking along the DNA between the enhancer and TSS
2. eRNAs themselves or their transcription allows enhancers to adopt an open chromatin configuration required for gene activation
Testing the eRNA transfer fuction
Placing transcription termination sequence between eRNA and target gene should block the transfer of RNA pol II
Issues with tracking model
eRNAs do not originate between the enhancer and TTS of associated gene
RNA pol II is delivered to TSS via DNA looping – eRNAs stabilise interactions between DNA and cohesion/mediator
Idea of open chromatin and eRNA
Enhancer in a closed chromatin state – enhancer inactive + gene inactive
Transcription at enhancer promotes an open chromatin state – enhancer becomes active
Enhancer promotes transcription of gene
Enhancer in a closed chromatin state – enhancer inactive
Block transcription elongation Histone marks not deposited Enhancer remains inactive
eRNAs interact with chromatin modifiers
eRNAs promote activity of CREB-binding protein (CBP) leading to increased acetylation of surrounding histone tails (open configuration)
Transcriptional activation via changes in chromatin landscape –enhancer/promoter
Interactions between eRNAs and chromatin modifying factors is non specific
eRNAs interact with transcriptional co-activators
eRNAs interact with bromodomain of co-activator BRD4 and both enhance and stabilize binding to nearby acetylation marks – maintains enhancer and gene activation
BRD4 binding to eRNA is locus specific – associates only with BRD4-bound enhancers - cis regulation
eRNA maintain the transcription factor
eRNAs capture disassociating transcription factors to enhance their occupancy at enhancers (TF trapping)
Generates a positive-feedback loop in which TFs stimulate local enhancer transcription, which increases occupancy – increases and stabilises gene expression
Backsplicing
