Transcript NisimNaim-AdiPotok

Final project –
Computational Biology
Identifying transcription
factories not involved in
pre-mRNA splicing
‫מר יהודה ברודי‬
‫טל‬-‫ד"ר ירון שב‬
:‫בהנחיית‬
‫ניסים נעים‬
‫עדי פוטוק‬
:‫מגישים‬
Introduction
There is evidence of transcription factories
which contain accumulations of RNA polymerase II.
Genes are moving towards the factories in order to be
transcribed.
Introduction
Splicing is a co-transcriptional modification of an mRNA,
in which introns are removed and exons are joined.
U1 and U2 are parts of the spliceosome machinery.
http://www.mpibpc.mpg.de/groups/pr/PR/2008/08_10_eng_RN/
Project goal
Identification and classification of
transcription factories, and categorizing the
factories by their probability to undergo
splicing.
RNA FISH
Fluorescence In-Situ Hybridization
Using a labeled oligonucleotide
probe to detect a specific
mRNA of interest.
Immunofluorescence
Using a fluorescent labeled
antibody to detect U1snRNA,
U1snRNA and RNA polymerase II.
mRNA
Wide-Field Microscopy
The cells are illuminated with light
of a certain wavelength and emit
light of a different wavelength.
This technique is used to acquire 3D
images of a specimen. Each image
is composed of several 2D layers.
Deconvolution
Problem: The light emitted from the fluorescent
molecules disperses, as the layer gets farther away
from the molecules.
Solution: this method is used to focus the light back
to its original source, in order to create an image more
similar to the original image.
Deconvolution
Before
After
Image analysis
IMARIS – Tool for analyzing images
 Wide graphical abilities.
 Embedded link to MATLAB programs.
Gene constructs: e1 and e3
•
e1 gene:
no splicing
•
e3 gene:
undergoes splicing
Splicing is co-transcriptional
Splicing factors can be identified at the site
of transcription
U1 snRNA
snRNAU1
MS2
MS2
Transcription
site
e1
U1 snRNA
MS2
U2 snRNA
snRNAU2
Transcription
site
U1 snRNA
U2 snRNA
snRNAU1
Nucleoplasm
U1 snRNA
U2 snRNA
snRNAU2
e3
Nucleoplasm
U2 snRNA
Splicing is co-transcriptional
snRNAU1
Splicing factors can be identified at the site
of transcription
RNA pol II Immunofluorescence
Cytoplasm
Nucleolus
Nucleoplasm
Transcription
factory
Step I – Identify Factories
•
Use “dynamic threshold” to intensify the areas with
high values, compared with their surroundings.
Dynamic
threshold
Intensity of
pixels in the
image
Step I – Identify Factories
Locate the centers of these areas
Step I – Identify Factories
Expand each center to the whole factory area
Step I – Identify Factories
Use the “find connected components” function
to differentiate between factories.
e3 transcription factory
LacI
mRNA
RNA Pol II
e3 transcription factory
mRNA molecules
surrounding the gene
RNA pol II and mRNA molecules
surrounding the gene
Step II – Calculate Correlation
•
Normalization of the U1 and U2 images.
•
Correction of pixel shift.
Step II – Calculate Correlation
•
We tried several methods to calculate correlation:
Pearson coefficient
average of U1 / average of U2
curve fit (aX + b)
Normalized count
Normalized count
Current results
U1 / U2
U1 / U2
Step III – classifying factories
•
The best method to divide the factories into two
distinct groups, is ….
•
Consensus correlation similar to that of e3 gene 
high probability of undergoing splicing.
•
Different / No consensus correlation 
low probability of undergoing splicing.
The final output
•
Using color gradient to color factories according to
the probability of undergoing splicing.
Biological conclusions
•
A few hundreds of transcription factories in each
nucleus, as mentioned in articles from recent years.
? Do factories tend to gather, or do they operate
throughout the nucleus.
? Do active factories concentrate in the center
of the nucleus.
What’s next?
•
Improve factory identification (more automatically).
•
Displaying each factory as an individual object
in IMARIS.
•
Analyze more images of e1 and e3 genes, to find
the differences between their factories.
•
Check the correlation of U1 and U4 factors.
Thanks


Dr. Yaron Shav-Tal
Mr. Yehuda Brody
http://io9.com/photogallery/scienceart2009/1008301614