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Promoters and expression
Transcriptome Promoters and expression
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The transcriptome transcriptome is the set of all RNA molecules Including: mRNA rRNA tRNA other non-coding RNA
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Measuring gene expression levels
Outline Measuring gene expression levels New understandings of promoters directionality My notes here
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Motivation Question: How can we measure protein levels in the body? The current way to measure was to look at genes expression levels But… New understandings show that we should measure each of the genes isoforms separately
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We would like to… Compare between expression levels of different genes Compare between expression levels of different isoforms of a single gene
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Different isoforms of one gene
Gene A Isoform 1 Isoform 2
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RNA-seq: reminder T C C G G A G T A
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What’s after RNA-seq? We will describe two different methods to approximate the gene expression Union count Intersect count
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counts reads falling on any of a gene’s exons
Union Count counts reads falling on any of a gene’s exons What could be the problems? Isoform 1 Isoform 2 Gene A Gene B
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Counts only reads on exons shard by all isoforms
Intersect Count Counts only reads on exons shard by all isoforms Isoform 1 Isoform 2 Gene A
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Two main problems How can we compare between isoforms of the same gene? What about the length of the gene?
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Cuffdiff 2 – Cole Trapnell
Change #1: Assign each fragment to a specific isoform Change #2: Normalize the number of fragments assigned to each isoform by its length
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Assigning fragments to isoforms
Basic Assumption: Percentage of fragments belonging to each isoform is uniform
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Assigning fragments to isoforms
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Cuffdiff 2 vs. old methods
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Cuffdiff 2 vs. old methods
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Cuffdiff 2 vs. old methods
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Cuffdiff 2 vs. old methods
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Measuring gene expression levels
Outline Measuring gene expression levels New understandings of promoters directionality My notes here
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Transcription as we know it
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Antisense upstream RNA
Antisense transcripts are transcribed from the strand opposite to that of the sense transcript It can be of either protein-coding or nonprotein- coding genes
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auRNA - Promoters Antisense upstream transcripts can arise from different kinds of promoters: Bidirectional promoters Cryptic promoters
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auRNA - Characteristics
Aside from their antisense orientation, they do not have unique features They lack of protein - coding potential Can contain specific domains that interact with DNA, RNA or proteins (like many ncRNAs) Many of them carry out specific functions
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So which strand is the downstream direction?
And where are those antisense transcripts?
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A new approach Christopher B. Burge Phillip A. Sharp MIT
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uaRNAs do not elongate efficiently
Two potential mechanisms: Inefficient release of paused RNAP 2 Early termination of transcription
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Early termination? It was found that upstream antisense region has a higher number of cleavage sites compared to the downstream sense sites
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Unique cleavage sites flanking TSS
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Cleavage sites – reminder
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The first step Determine whether members of the canonical cleavage machinery bind to uaRNAs Ten 3’ end processing factors were analyzed to find protein – RNA interactions (CLIP method)
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Specific binding of all ten factors was detected at uaRNA cleavage sites
The positional profiles identical or very similar to that of mRNA cleavage sites
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Understanding the molecular mechanism
So, how come there are 2 times the cleavage areas in the upstream direction?
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Understanding the molecular mechanism
Examine PAS frequencies!
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33% depletion of PAS downstream of TSS
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Sense genes exhibit large amount of U1 sites
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The U1-PAS model U1 snRNP complex suppresses cleavage and polyadenylation near a U1 site
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The U1-PAS model
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auRNA – Mechanisms of gene regulation
We will discuss the different steps: Effects on transcription initiation Effects on transcription elongation Post transcriptional effects
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Transcription initiation
LUC7L produces an antisense transcripts This transcript overlaps with HBA1 It methylates its promoter HBA1’s expression is silenced
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Transcription elongation
A collision can occur between two RNA polymerases Can lead to alternative isoforms (independent from splicing mechanisms)
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Post transcriptional The antisense transcript of Uchl1 binds to the 5’ UTR of the sense transcript The SINEB2 domain of the antisense molecule increases Uchl1 translation efficiency Two differences from previous examples: Positive effect Mechanism after the antisense is transcribed
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Post transcriptional
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Summary New technologies enable us to further understand genes expression levels Transcription process is not as “simple” as we thought Every transcript has its role
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Questions ?
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