Journal: Nucleic Acids Research

Article Title: The yeast phosphofructokinase β-subunit has RNA unwinding activity and modulates cell cycle progression

doi: 10.1093/nar/gkag184

Figure Lengend Snippet: Pfk1p and Pfk2p bind to functionally related RNAs in vivo . ( A ) Immunoblot analysis of RIC eluates. Top: Pfk1:TAP and Pfk2:TAP detected with Peroxidase-Anti-Peroxidase Soluble Complex (PAP) reagent. Middle: endogenous Pfk1p and Pfk2p proteins detected with Pfk antibodies; Tal1:TAP is as unconventional RBP used as positive control . Bottom: Endogenous Pfk1p and Pfk2p detected in pfk2Δ and pfk1∆ cells, respectively. Scp160p is an RBP control; Act1p is non-RBP negative control. Input refers to cell extracts; poly(A) refers to the addition of excess competitor polyadenylic acids. ( B ) Heatmap representation of the abundance of 1249 fRIP selected Pfk1p and Pfk2p RNA targets. Columns refer to independent Pfk1:TAP, Pfk2:TAP, and untagged mock control fRIPs; rows denote individual transcripts. The white-blue colour bar represents normalized read counts for respective transcripts. ( C ) Venn diagram showing overlap of Pfk1p and Pfk2p mRNA targets. The P- value (hypergeometric test) relates to the significance of overlap. ( D ) Overrepresented GO terms among 671 common Pfk1p and Pfk2p RNA targets. The circle diameter is proportional to the number of RNA targets, the colour refers to the −log 10 FDR. X -axis specifies the enrichment score. GO categories: BP, biological process; MF, molecular function. ( E ) Agarose gel showing products from reverse transcriptase-polymerase chain reaction (RT-PCR) reactions for detection of cell cycle related mRNA targets in fRIP eluates as marked in panel (B). Actin ( ACT1 ) is a negative control, PFK2 mRNA was previously shown to associate with Pfk2p .

Article Snippet: Kits (in order of appearance): Q5 ® Hot Start High-Fidelity 2× Master Mix (New England Biolabs, M0494S), QIAquick polymerase chain reaction (PCR) Purification Kit (Qiagen, 28104), Kinase-Ligase-DpnI (KLD) enzyme mix (New England Biolabs, M0554S), LR Clonase II plus kit (Thermo Fisher, 12538120), Q5 site-directed Mutagenesis kit (NEB, E0554S), DynabeadsTM mRNA DIRECTTM Purification kit (Thermo Fisher, 61011), PierceTM bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher, 23227), TURBO DNA-free kit (Thermo Fisher, AM1907), Ribo-Zero Gold ribosomal RNA (rRNA) Removal Kit (Illumina, MRZY1306), SensiFast complementary DNA (cDNA) synthesis kit (Meridian Bioscience, BIO-65053), SensiFast SYBR lo-ROX mix (Meridian Bioscience, BIO-94005), NEBNext ® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, E6111S), Bioanalyzer DNA high sensitivity kit (Agilent, 5067-4626); JetSeq DNA library preparation kit (Meridian Bioscience, BIO-68025), MyTaq Red Mix (Meridian Bioscience, BIO-25044), TMTpro 16-plex Label Reagent Set (Thermo Fisher, A44520 ), Biotin RNA Labelling Mix (Roche, 11685597910), Quick RNA Microprep kit (Zymo Research, R1050).

Techniques: In Vivo, Western Blot, Positive Control, Control, Negative Control, Agarose Gel Electrophoresis, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction