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'Role of CNOT1 protein in mRNA deadenylation' - BIOC 396 Undergraduate Research Project Application Form.

INSTRUCTIONS - PROFESSORS: Please print and review this form. Complete or correct the sections, as applicable, from "Supervisor's Name" to "Ethics, safety, and training". Please sign and date near the bottom ("Supervisor's signature").

INSTRUCTIONS - STUDENTS: You may receive this form by email, or you may download it from www.mcgill.ca/science/research/ours/396 after it has been posted. Either way, print and review this form. Complete or correct the sections, from "Student's Name" to "Student's Level", and sign ("Student signature). Ask your supervisor to sign her/his section near the bottom. Take it to the department corresponding to the course number; this may or may not be your own department. Do not register for a '396' course on Minerva until you receive departmental permission. Have a discussion with your supervisor about time/work expectations, keeping in mind that this is a 3-credit course (roughly, 10 hours per week for 12 weeks). Remember that a '396' course is an elective.

INSTRUCTIONS - DEPARTMENTS: After the unit chair/director/designate approves (or not) this project, please notify student. If approved, please give student permission to register on Minerva, and fax this form (with signatures) to the Office for Undergraduate Research in Science at 514-398-8102. The Office for Undergraduate Research in Science will later post the project online at www.mcgill.ca/science/research/ours/396/listing, indicating whether the project is open for students to apply or taken.

QUESTIONS OR FEEDBACK? Contact Victor Chisholm by email, or phone 514-398-5964

Supervisor's Name: Nahum Sonenberg and Alexey Karetnikov

Supervisor's Email: alexey [dot] karetnikov [at] mail [dot] mcgill [dot] ca

Supervisor's Phone: 438-885-3125 (A.K.)

Supervisor's Website:

Supervisor's department: Biochemistry

Course number: BIOC396 (Biochemistry)

Term: Fall 2011-2012

Project start date: Wednesday, September 1, 2011

Project end date: Tuesday, December 6, 2011

Project title: Role of CNOT1 protein in mRNA deadenylation

Project description:

mRNA deadenylation is one of the crucial steps of mRNA metabolism and regulation of gene expression (1). Deadenylation is performed by several classes of deadenylase protein complexes (1, 2). CCR4-Not1 complex is a highly conserved protein ensemble containing deadenylases of two types - 1) from DEDD family (CNOT7 and CNOT8 subunits in human), and 2) from EEP family (CNOT6 and CNOT6L in human) (2). Molecular mechanism of functioning of the CCR4-Not1 complex remains largely unclear. Human CCR4-Not1 has been shown to deadenylate mRNA associated with microRNA (miRNA)-induced silencing complex (miRISC) (3). miRNAs repress translation and promote mRNA deadenylation by targeting 3' untranslated regions (4-8). GW182 protein, one of the key components of miRISC, recruits CCR4-Not1, triggering efficient deadenylation (9, 10). Interaction between GW182 and the poly(A)-binding protein is also essential for deadenylation, and it has been hypothesized to position CCR4-Not1 in close vicinity of the poly(A) tail (9, 10). In addition, CCR4-Not1 has been shown to repress translation independent of deadenylation (11).

CNOT1, the largest, scaffolding subunit of the CCR4-Not1 complex, interacts with most of the other subunits (2). The middle part of human CNOT1, containing amino acid residues 727-1449, mediates CNOT1 binding to CNOT7 (12). In yeast NOT1, the middle amino acid region (positions 667-1152) interacts with catalytic subunits CCR4 and CAF1, whereas the C-terminal part (residues 1490-2108) interacts with NOT2, NOT4 and NOT5 (13). Our preliminary data suggest that the silencing domain of GW182 interacts with human CCR4-Not1 through its CNOT1 subunit. However, it is not known which part of CNOT1 participates in this interaction. One of the goals of this project is to map CNOT1 determinants essential for binding to GW182. To this end, we will divide the CNOT1 gene to six overlapping fragments and clone each gene fragment into the pGEX6p1 plasmid, followed by induction of protein overexpression in E. coli. Each CNOT1 fragment will be expressed as a fusion protein, containing N-terminal GST and T7 tags, as well as a C-terminal 6xHis tag. Proteins will be purified from E. coli cells by using GST-glutathione affinity chromatography followed by 6xHis-Ni2+-NTA chromatography. The T7 tag will be used for detecting proteins by Western blotting. Purified CNOT1 fragments will be used for in vitro pull-down assays with the silencing domain of GW182 (the latter will be MBP-tagged and purified by using MBP-amylose affinity chromatography). Also, we will perform CNOT1 knock-down in HeLa cells by using a shRNA-expressing lentiviral vector, and will overexpress the CNOT1 fragments in these cells. Cell lysate will be used in similar pull-down experiments to detect endogenous proteins binding to the CNOT1 fragments. In addition to GW182, we will test whether these CNOT1 fragments bind translation initiation factors. Considering that CCR4-Not1 is able to repress translation independent of deadenylation (11), this will allow us to determine CNOT1 regions responsible for this deadenylation-independent translational repression. Finally, based on the sequence differences between CNOT1 from human and NOT1 from yeast, which does not have miRNA pathway, we will delineate CNOT1 regions involved in miRNA-dependent and miRNA-independent deadenylation.

Our study will provide further insight into molecular mechanisms underlying functions of the CCR4-Not1 deadenylase complex in regulating gene expression.

References: (1) Wiederhold & Passmore (2010) Biochem Soc Trans 38:1531-1536 (2) Bartlam & Yamamoto (2010) Protein Cell 1:443-452 (3) Piao et al (2010) Mol Cell Biol 30:1486-1494 (4) Mathonnet et al (2007) Science 317:1764-1767 (5) Eulalio et al (2009) RNA 15:21-32 (6) Fabian et al (2010) Annu Rev Biochem 79:351-379 (7) Huntzinger & Izaurralde (2011) Nat Rev Genet 12:99-110 (8) Djuranovic et al (2011) Science 331:550-553 (9) Fabian et al (2009) Mol Cell 35:868-880 (10) Zekri et al (2009) Mol Cell Biol 29epr:6220-6231 (11) Cooke et al (2010) J Biol Chem 285:28506-28513 (12) Sandler et al (2011) Nucleic Acids Res 39:4373-4386 (13) Bai et al (1999) Mol Cell Biol 19:6642-6651

Prerequisite: 1 term completed at McGill + CGPA of 3.0 or higher; or permission of instructor.

Grading scheme (The final report must be worth at least 50% of final grade): Final grade shall be based on laboratory performance as evaluated by the research supervisor (50%) and the final written research report (minimum 10 pages) graded by the supervisor and the course coordinator or the coordinator's delegate (50%)

Project status - This project is: Taken. The professor has no more '396' projects this term.

How students can apply: N/A; this project is filled.

Ethics, safety, and training: Supervisors are responsible for the ethics and safety compliance of undergraduate students. This project involves:

  • Biohazardous substances
  • Handling chemicals

Student's Name:

Student's McGill ID:

Student's Email (first [dot] last [at] mail [dot] mcgill [dot] ca):

Student's Phone:

Student's Program:

Student's Level (U0 / U1 / U2 / U3):

Student's signature - I have not applied for another '396' course in this term:

Supervisor's signature - I give my permission for the student identified above to register for this project under my supervision:

Date:

Unit chair/director/designate's name:

Unit chair/director/designate's signature - I certify that this project conforms to departmental requirements for 396 courses:

Date: