1. Promoters and expression
Transcriptome
Promoters and expression

2. The transcriptome
transcriptome is the set of all RNA molecules
Including:
mRNA
rRNA
tRNA
other non-coding RNA

3. Measuring gene expression levels
Outline
Measuring gene expression levels
New understandings of promoters directionality
My notes here

4. 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

5. We would like to…
Compare between expression levels of different genes
Compare between expression levels of different isoforms of a single gene

6. Different isoforms of one gene
Gene A
Isoform 1
Isoform 2

7. RNA-seq: reminder T C C G G A G T A

8. What’s after RNA-seq?
We will describe two different methods to approximate the gene expression
Union count
Intersect count

9. 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

10. 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

11. Two main problems
How can we compare between isoforms of the same gene?
What about the length of the gene?

12. 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

13. Assigning fragments to isoforms
Basic Assumption: Percentage of fragments belonging to each isoform is uniform

14. Assigning fragments to isoforms

15. Cuffdiff 2 vs. old methods

16. Cuffdiff 2 vs. old methods

17. Cuffdiff 2 vs. old methods

18. Cuffdiff 2 vs. old methods

19. Measuring gene expression levels
Outline
Measuring gene expression levels
New understandings of promoters directionality
My notes here

20. Transcription as we know it

21. 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

22. auRNA - Promoters
Antisense upstream transcripts can arise from different kinds of promoters:
Bidirectional promoters
Cryptic promoters

23. 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

24. So which strand is the downstream direction?
And where are those antisense transcripts?

25. A new approach
Christopher B. Burge
Phillip A. Sharp
MIT

26. uaRNAs do not elongate efficiently
Two potential mechanisms:
Inefficient release of paused RNAP 2
Early termination of transcription

27. Early termination?
It was found that upstream antisense region has a higher number of cleavage sites compared to the downstream sense sites

28. Unique cleavage sites flanking TSS

29. Cleavage sites – reminder

30. 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)

31. 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

32. Understanding the molecular mechanism
So, how come there are 2 times the cleavage areas in the upstream direction?

33. Understanding the molecular mechanism
Examine PAS frequencies!

34. 33% depletion of PAS downstream of TSS

35. Sense genes exhibit large amount of U1 sites

36. The U1-PAS model
U1 snRNP complex suppresses cleavage and polyadenylation near a U1 site

37. The U1-PAS model

39. auRNA – Mechanisms of gene regulation
We will discuss the different steps:
Effects on transcription initiation
Effects on transcription elongation
Post transcriptional effects

40. Transcription initiation
LUC7L produces an antisense transcripts
This transcript overlaps with HBA1
It methylates its promoter
HBA1’s expression is silenced

41. Transcription elongation
A collision can occur between two RNA polymerases
Can lead to alternative isoforms (independent from splicing mechanisms)

42. 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

43. Post transcriptional

44. 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

45. Questions ?