Puf Family role in yeast cells

RBPs - RNA-binding proteins are involved in different aspect of post-transcriptional gene regulation. The Puf in S. cerevisiae are structurally related family of cytoplasmatic RBPs. The proteins got several consecutive repeats of the Pumilio homology domain (Pum-HD). Bind to the 3'UTR sequence. The five members bear 6-8 Puf (Pum-HD) repeats.

Your mission, should you choose to accept it, involves identify specific mRNAs that interact with the five members of the Puf family. Finding if there is a consensus sequence in the mRNAs that binds by those Puf members.

ENDS

milestone open Previous Milestones

milestone open Background Research

Daniel Moore

To summarise what we already know:

milestone closed TAP-tag the Puf family

Daniel Moore

Based on the Rigaut et al. 1999 - built a 5 contracts with a sequence encoding two IgG-binding units of protein A, a specific protease recognition site, and a calmodulin-binding domain, was fused in-frame at the C-terminus of the respective open reading frame (ORF)in its original chromosomal location.

milestone open Current Milestones

milestone open Isolate Puf Proteins

Daphne Rosenfeld

Will isolate Puf Proteins.

  • Transfection

milestone open Extract cells, recover mRNA and labelling

Daniel Moore

Harvest the cells and extract the ribonucleoprotein complexes (IgG binding) RNA isolation from immunopurified samples. Labelling with fluorescent dyes

milestone open The mobility of GFP-tagged Puf1

Daphne Rosenfeld 03/28/2012

The fluorescence of a GFP-tagged protein is bleached in the region of interest.

milestone open Subcellular distribution of Puf proteins

Daniel Moore

Localization of the TAP-tagged Puf proteins with immunofluorescence with antibodies against the TAP tag.

+ New Milestone