Transcriptome-wide profiling of Yeast Phosphofructokinase 1 and 2 RNA targets
The RNA targets of the yeast Pfk1 and Pfk2 were mapped on a transcriptome scale using formaldehyde-assisted crosslinking and RNA-protein immunoprecipitation followed by high throuput sequencing (fRIP-seq). Tandem affinity purification (TAP) tagged Pfk1 and Pfk2 were usesd to capture the transcritps bound by Pfk1 and Pfk2. We also included a mock IP control i.e. wild type treated exactly the same way as the fRIP-seq samples to account for non-specific RNA interactions with the beads. Moreover, we ran total RNA-seq on wild type control for data normalization. We found that Pfk1 and Pfk2 bind mainly to mRNAs that code for proteins invovled in distinct functions . Our data suggests that, besides their well-characterized functions as glycolytic enzymes, they perfom additional functions as RNA-binding involved in posttranscriptional regulation of gene expression. Overall design: Our aim was to map transcripts bound by the yeast Pfk1 and Pfk2 to explore their RNA-bining functions and to obtain and insight into their potential secondary function as posttranscriptional regulators of gene expression. We performed formaldehyde assisted crosslinking followed by RNA-protein immuniprecipitation (fRIP-seq) to identify RNAs bound by Pfk1 and Pfk2 under normal physiological conditon. Diploid yeast cells expressing either Pfk1 or Pfk2 with Tandem Affinity purification (TAP) tags as well as dipliod wild type cells for mock IP control were grown in rich YPD media to mid-log phase (OD600 = 0.6) then crosslinked with low concentration of formaldehyde to a final concentration of 0.3% followed by quenching of crosslinking using glycine (final concentration 125 mM). Cells were pelleted then washed three times with 1X PBS then flash frozen in liquid nitroen. Frozen cell pellets were ground under cryogenic conditions using mortar and pestel to fine powders then powders were resuspended in fRIP lysis buffer. Pfk1:TAP-RNA and Pfk2:TAP-RNA were captured using Pan mouse IgG beads. Crosslinking was reveresed followd by proteinase K digestion of proteins then RNA was extracted using standard acidic phenol method. RNA was then converted to cDNA followed by second strand synthesis then double-stranded cDNA was sheared using sonication. Sequencing libraries were constructed using Bioline JetSeq libraray preparation kit as described in product manual. We also perfomed total RNA-seq from wild type diploid cells for data normalization. Samples were sequenced on Illumina HiSeq2500 with minimum of 5 million reads per library. Our data showed that both Pfk1 and Pfk2 bind to hunderds on mRNAs coding for proteins involved in various cellular functions. Interestingly, our data suggests that Pfk1 and Pfk2 bind to mRNAs coding for proteins involved in distinct functions indicating that the two proteins are involved in different cellular functions as posttranscriptional regulators.