cufi 1  (ATCC)

Journal: bioRxiv

Article Title: La protein binding to telomerase RNA supports an evolutionary relationship between plant and ciliate telomerase pathways

doi: 10.64898/2026.01.19.700320

Figure Lengend Snippet: ( A ) Domain organisation of La and LARP7 proteins ( , , ), Tetrahymena Mlp1 is based on Alphafold3 domain predictions and sequence alignments (Figure S5A) and includes an apparent additional xRRM in a region known to bind RNA . Domains are as annotated, ‘+’ or ‘-‘ indicates regions of the sequence with correspondingly charged residues at neutral pH. ( B ) AtLa1 EMSA using 1% agarose gel with 80 nM AtTR 1-268 and protein concentrations (nM) as annotated, visualized by fluorescent staining. ( C ) Representative AtLa1 EMSAs with pre-tsnoR43.1, pretsnoR43.1ΔU and snoR43.1 substrates ( , ), as annotated, conditions as (B). White lines have been added to separate panels from the same gel. ( D ) ELISA experiments detecting immunolabelled AtLa1 bound to ≤0.5 pmol biotinylated AtTR 1-268 and non-biotinylated competitor RNA as annotated, (ytRNA, budding yeast tRNA). Lines show equations fit to data used to calculate K D , average AtTR values are shown in all experiments for comparison, error bars are the standard deviation. ( E ) ELISA experiments as (D) with plant TR competitors (full details in Table S2). ( F ) Comparison of AtLa1-AtTR binding assays used in this work. Solid black circles are average initial capillary fluorescence values (upper panel) or 1.5 s T-jump data (lower panel) from MST experiments, signal is from 5 nM 3’ Cy5-labelled AtTR 1-268. Solid lines show equations fit to data used to calculate K D (Table S3), dotted lines for both fast processes are Hill equation fits with n=2 for comparison, error bars are standard deviation. Open black circles with dashed lines are normalised fluorescence emission at 680 nM from 20 nM AtTR-Cy5, with excitation at 625 nM using a traditional spectrofluorometer (full spectra in Fig. S1H). Closed red circles show normalised Δε at 265 nM from circular dichroism experiments monitoring 50 nM unlabelled AtTR (difference spectra in Fig. S1I). Blue closed circles are normalised fractional saturation of 40 nM unlabelled AtTR calculated from band shift distances for EMSAs (Fig. S1J) and open blue circles and dotted lines are 1/normalised signal change for ELISA competition experiments (derived from (D) for comparison). For all experiments other than ELISAs, AtLa1 concentrations are divided by the fold increase in AtTR concentration. ( G ) MST experiments detecting AtTR-Cy5 in the presence of excess unlabelled competitor RNA as annotated (Table S4), AtTR-AtLa1 data are reproduced from (F) for comparison.

Article Snippet: The supernatant was applied to SpinTrap GST or Ni Sepharose spin columns (Cytiva, #28-4013-53, #28-9523-59), reused and periodically refilled with 0.4 mL 50% Glutathione Sepharose® 4 Fast Flow (Cytiva,# 17-5132-01) or Ni-NTA agarose Fast Flow resin (Cube Biotech, # 31105).

Techniques: Sequencing, Agarose Gel Electrophoresis, Staining, Enzyme-linked Immunosorbent Assay, Comparison, Standard Deviation, Binding Assay, Fluorescence, Circular Dichroism, Electrophoretic Mobility Shift Assay, Derivative Assay, Concentration Assay

Journal: bioRxiv

Article Title: La protein binding to telomerase RNA supports an evolutionary relationship between plant and ciliate telomerase pathways

doi: 10.64898/2026.01.19.700320

Figure Lengend Snippet: ( A ) Domain structure of AtLa1, including LaM (grey) and xRRM (red), as predicted by Alphafold2 , and visualized using ChimeraX . xRRM residues chosen for mutagenesis studies are shown in a magnified panel (the same colour scheme is used in B-E). ( B-E ) Binding of AtLa1 fragments (B, C) and AtLa1 xRRM point variants (D, E) to AtTR investigated by EMSA (B, D) and MST (C, E). (B, D) Representative band shift of 80 nM AtTR 1-268 caused by binding of AtLa1 protein constructs (concentrations in nM) is visualised in agarose gel by fluorescent staining. White lines have been added to separate panels in the same gel (full image in Figure S2). (C, E) Initial capillary fluorescence values from MST experiments (upper panels) or MST 1.5 s T-jump data (lower panels) for 3’ Cy5-labelled AtTR 1-268 and La1 fragments as indicated, with average AtLa1 FL data from reproduced for comparison. Open symbols and dashed lines in (C), lower panel only, show data in the presence of 100x excess unlabelled tRNA competitor. Lines show equations fit to data used to calculate K D , error bars are the standard deviation.

Article Snippet: The supernatant was applied to SpinTrap GST or Ni Sepharose spin columns (Cytiva, #28-4013-53, #28-9523-59), reused and periodically refilled with 0.4 mL 50% Glutathione Sepharose® 4 Fast Flow (Cytiva,# 17-5132-01) or Ni-NTA agarose Fast Flow resin (Cube Biotech, # 31105).

Techniques: Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Construct, Agarose Gel Electrophoresis, Staining, Fluorescence, Comparison, Standard Deviation