1. Final project – Computational Biology
Identifying transcription factories not involved in pre-mRNA splicing
בהנחיית: מר יהודה ברודי
ד"ר ירון שב-טל
מגישים: ניסים נעים
עדי פוטוק

2. 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.

3. 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.

4. Project goal
Identification and classification of transcription factories, and categorizing the factories by their probability to undergo splicing.

5. Fluorescence In-Situ Hybridization
RNA FISH
mRNA
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.

6. 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.

7. 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.

8. Deconvolution
Before
After

9. Image analysis IMARIS – Tool for analyzing images
Wide graphical abilities.
Embedded link to MATLAB programs.

10. Gene constructs: e1 and e3
e1 gene:
no splicing
e3 gene:
undergoes splicing

11. Splicing factors can be identified at the site of transcription
Splicing is co-transcriptional
Splicing factors can be identified at the site of transcription
snRNAU2
U2 snRNA
snRNAU1
U1 snRNA
MS2
MS2 e3
Transcription site
U1 snRNA
Nucleoplasm
snRNAU1
snRNAU2
U2 snRNA
U2 snRNA
U1 snRNA
MS2
Transcription site e1
U1 snRNA
Nucleoplasm
U2 snRNA

12. Splicing factors can be identified at the site of transcription
Splicing is co-transcriptional
Splicing factors can be identified at the site of transcription
snRNAU1

13. RNA pol II Immunofluorescence
Cytoplasm
Nucleolus
Nucleoplasm
Transcription factory

14. 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

15. Step I – Identify Factories
Locate the centers of these areas

16. Step I – Identify Factories
Expand each center to the whole factory area

17. Step I – Identify Factories
Use the “find connected components” function
to differentiate between factories.

18. e3 transcription factory
LacI
mRNA
RNA Pol II

19. e3 transcription factory
RNA pol II and mRNA molecules surrounding the gene
mRNA molecules surrounding the gene

20. Step II – Calculate Correlation
Normalization of the U1 and U2 images.
Correction of pixel shift.

21. Step II – Calculate Correlation
We tried several methods to calculate correlation:
Pearson coefficient
average of U1 / average of U2
curve fit (aX + b)

22. Current results
Normalized count
Normalized count
U1 / U2
U1 / U2

23. 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.

24. The final output
Using color gradient to color factories according to the probability of undergoing splicing.

25. 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.

26. 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.

27. Thanks Dr. Yaron Shav-Tal Mr. Yehuda Brody