RNA polymerase II depletion promotes transcription of alternative mRNA species Authors: Lijian Yu, Mayuri Rege, Craig L. Peterson and Michael R. Volkert1 Affiliation: Microbiological and Physiological Systems, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA BMC Molecular Biology Article accesses: 1193 DOI 10.1186/s12867-016-0074-8 Received: 13 January 2016 | Accepted: 18 August 2016 | Published: 30 August 2016
Transcript
RNA polymerase II depletion promotes
transcription of alternative mRNA species
Authors: Lijian Yu, Mayuri Rege, Craig L. Peterson and Michael R. Volkert1
Affiliation: Microbiological and Physiological Systems, University of Massachusetts
Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
Program in Molecular Medicine, University of Massachusetts Medical
School, 373 Plantation Street, Worcester, MA 01605, USA
BMC Molecular Biology
Article accesses: 1193
DOI 10.1186/s12867-016-0074-8
Received: 13 January 2016 | Accepted: 18 August 2016 | Published: 30 August 2016
RNA polymerase II depletion promotes
transcription of alternative mRNA species
Course Code: GEB202
Course Title: Molecular Biology
Course Instructor: “Abid Al Reza”
Lecturer, Department of Genetic Engineering and Biotechnology,
East West University.
Presented By:
1. Richita Islam
2. Nayeem Dewan
3. Bitali Islam
4. Md. Shabab Mehebub
Keywords• Transcriptional stress: Transcription machinery may play an important role in
sensing DNA damage and activating DNA repair and stress response pathways
when stalled at blocking lesions.
• RNA polymerase depletion: means the inactivation of RNA polymerase.
• Altered polyadenylation: is emerging as a widespread mechanism used to
control gene expression.
• mRNA induction: relief of repression for mRNa under negative control by a
repressor.
• Rapamycin: used in biology research as an agent for chemically induced
dimerization.
• Tbp1: TATA binding protein 1
• ChIP: chromatin immunoprecipitation
Background of the Research
• Cells respond to numerous internal and external stresses, such as
heat, cold, oxidative stress, DNA damage, and osmotic pressure
changes.
• In most cases, the primary response to stress is transcriptional
induction of genes that assist the cells in tolerating the stress and
facilitate the repair of the cellular damage.
• However, when the transcription machinery itself is stressed,
responding by such standard mechanisms may not be possible.
Introduction
The RPB2 gene locus has served as an ideal system to investigate
effects of UV damage in the cell.
The arrest of multiple RNA polymerase complexes at DNA damage
sites the genome also reduces the number of functionally active and
free, RNA polymerase molecules.
Te arrest of RNAP II initiates a DNA repair response known as
transcription coupled repair (TCR).
UV treatment generally inhibits transcription genome-wide until the
damage is repaired.
Objectives
To test whether production of the RPB2 long form is a general hallmark of transcriptional stress.
To find depletion of RNAPII triggers transcriptional changes similar to the changes seen after UV
treatment.
To examine transcriptional stress that triggers induction of specific genes and modulation of
polyadenylation (polyA) site usage.
Methods
1• Yeast strains and plasmids
2• Anchor-away and UV irradiation
3• Heat shock at 37 °C
4• Northern blot analysis
5• RT-PCR analyses of RNA
6• Analysis of the DRS dataset
7• ChIP experiments
Result and Discussion
Depletion of RNA polymerase II induces the long form
of RPB2 mRNA
Fig. 1 Polymerase stress increases transcription of the long RPB2 mRNA.
Induction of the long form of RPB2 is not due to increased
RNAPII occupancy
Fig. 2: Chromatin Immunoprecipitation of RNA polymerase subunits at the RPB2 locus after RNAPII depletion.
To ensure that it doesn’t used
reserve polymerase
Depletion of RNAPI promotes expression of the long form of RBP2
Fig. 3: Depletion of RNAPI
but not RNAPIII induces the
long RPB2 mRNA.
Analysis of genome wide changes in transcription
after RNAPII depletion
Fig. 4: Analysis of Direct RNA Sequencing (DRS) data for mRNA isoforms after RNAPII depletion.
Analysis of genome wide changes in transcription after RNAPII
depletion
Fig. 5: Depletion of RNAPI or RNAPII induces expression of a subset of mRNAs.
Conclusion
This research demonstrated that depletion of RNAPII and RNAPI,
but not RNAPIII results in a transcriptional stress response that
triggers the induction of specific mRNAs.
Finally mRNAs induced also exhibit a change in the
polyadenylation site.
Transcriptional stress response must, result in modification of the
