Journal: bioRxiv

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

doi: 10.1101/2025.02.01.636022

Figure Lengend Snippet: (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 Pfk2 proteins detected with Pfk antibodies; Tal1:TAP is as non-conventional RBP . Bottom: Endogenous Pfk1p and Pfk2p detected in pfk1Δ and pfk2Δ cells. Scp160p is a RBP control; Act1p is non-RBP negative control. Input refers to cell extracts; poly(A) designates the addition of excess competitor polyadenylic acids. (B) Heatmap representation of the abundance of 1,249 fRIP selected Pfk1p and Pfk2 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 normalised read counts for respective transcripts. (C) Venn diagram showing overlap of Pfk1p and Pfk2 mRNA targets. The p -value (hypergeometric test) relates to the significance of overlap. (D) Selection of GO terms overrepresented among 671 common Pfk1p and Pfk2 RNA targets. The diameter of the circle is proportional to the number of RNA targets, the colormap 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 RT-PCR reactions for detection of cell cycle related target mRNAs in fRIP eluates as marked in (B). Actin ( ACT1 ) is a negative control. PFK2 was previously shown in association with Pfk2p 5 .

Article Snippet: The CDSs were then subcloned into the pDEST17 gateway vector (ThermoFisher, 11803012) using LR clonase II kit to generate pDEST17-Pfk1 and pDEST17-Pfk2 expression vectors (ThermoFisher, 12538120).

Techniques: Western Blot, Control, Negative Control, Selection, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

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

doi: 10.1101/2025.02.01.636022

Figure Lengend Snippet: (A) Experimental design for glucose (glc) recovery experiments. Cells were grown in YPD (+G), then starved in media lacking glc for 20 min (-G) and recovered upon re-addition of glc for 20 min (R). (B) Immunoblot of input extracts (left) and RIC eluates (right) from glc recovery experiments at indicated stages (+G, -G, R). Monitored proteins are labelled to the right; a molecular weight marker is indicated to the left. (C) Polysomal absorbance profiles of pfk1Δ and pfk2Δ cells grown in YPD medium are shown at the top. Fractions numbers are indicated and those containing polysomes are highlighted in purple. Immunoblot analysis of fractions monitoring the distribution of Pfk1p and Pfk2p; and upon treatment of extracts with 30 mM EDTA to dissociate ribosomes (see Methods; Figure S3) are given below. Rpl35p is a ribosomal protein of the large subunit, Act1p is a non-ribosomal associated control protein. (D) Distribution of CLN3 , BUB3 and ACT1 mRNA levels across sub-polysomal and polysomal fractions (purple) obtained from wild-type (wt; grey), pfk1Δ (blue) and pfk2Δ (red) cells. RNA was isolated from each fraction and quantified by RT-qPCR. The y -axis denotes mRNA levels in each fraction calculated as the percentage of the total (mean values ± stdev, n=3). (E) BUB3 , and CLN3 mRNA levels relative to ACT1 levels determined by RT-qPCR in total RNA isolated from wt, pfk1Δ and pfk2Δ cells (mean values ± stdev, n=3). * p < 0.05 (student’s t -test).

Article Snippet: The CDSs were then subcloned into the pDEST17 gateway vector (ThermoFisher, 11803012) using LR clonase II kit to generate pDEST17-Pfk1 and pDEST17-Pfk2 expression vectors (ThermoFisher, 12538120).

Techniques: Western Blot, Molecular Weight, Marker, Control, Isolation, Quantitative RT-PCR

Journal: bioRxiv

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

doi: 10.1101/2025.02.01.636022

Figure Lengend Snippet: (A) Venn Diagram displaying the overlap of numbers of proteins with altered levels (FDR ≤ 10%) in the mutant compared to wild-type (wt) cells. (B) Heatmap displaying a subset of GO terms (rows) enriched among the proteins with increased (upright arrow) or reduced (downward arrow) levels in indicated mutants (columns). The colour intensity corresponds to Bonferroni corrected FDRs. (C) Boxplots depicting relative fold-changes of protein levels in pfk1Δ (blue), pfk2Δ (yellow) and map1Δ (grey) cells compared to wt cells (log 2 scale). Whiskers extend from the 10 th to the 90 th percentile. The distribution of all proteins is shown at the top. GO terms are indicated to the left and the number of plotted Pfk2p mRNA target encoded proteins within the respective GO group is indicated in brackets. Asterisks refer to p -values determined in a student’s t -test with Welch’s correction comparing the distribution of Pfk2p mRNA target encoded proteins assigned to specified GO term with the distribution of all measured features: *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: The CDSs were then subcloned into the pDEST17 gateway vector (ThermoFisher, 11803012) using LR clonase II kit to generate pDEST17-Pfk1 and pDEST17-Pfk2 expression vectors (ThermoFisher, 12538120).

Techniques: Mutagenesis

Journal: bioRxiv

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

doi: 10.1101/2025.02.01.636022

Figure Lengend Snippet: The enzymatic Pfk1-Pfk2 protein complex is shown to the left as a tetramer for simplicity; allosteric adenosine phosphate binding is indicated only (ATP required for catalysis is not shown). The R-state refers to high enzyme activity; the T-state to low activity. Cellular energy levels are indicated with a colour bar (AMP/ADP - ATP levels) to the left. Mg-ATP binding enables Pfk2p-RNA and ribosome interactions enhancing mRNA translation, possibly promoting cell-cycle progression (shown to the right). Parts created with BioRender.com.

Article Snippet: The CDSs were then subcloned into the pDEST17 gateway vector (ThermoFisher, 11803012) using LR clonase II kit to generate pDEST17-Pfk1 and pDEST17-Pfk2 expression vectors (ThermoFisher, 12538120).

Techniques: Binding Assay, Activity Assay