Human hnRNPA1 reorganizes telomere-bound replication protein A

Abstract

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection.

Graphical Abstract

Figure 1.

DBD-A of RPA^E240K displays altered conformational dynamics. (A) Single-molecule TIRFM assay for monitoring the conformational dynamics of RPA. Biotinylated ssDNA molecules (dT100) are tethered to the surface of the smTIRFM flow cells. Binding of the MB543-labeled RPA manifests in the appearance of the fluorescence signal, while conformational dynamics of the DNA-bound RPA manifests in changes in the fluorescence intensity. (B) Experimental scheme. (C) A representative fluorescence trajectory (time-based changes in the MB543 fluorescence in a specific location in the smTIRFM flow cell) for the wild type RPA labeled with MB543 at the DBD-A (RPA-DBD-A^MB543; green) overlaid with an idealized trajectory (black) obtained by globally fitting all trajectories to a four-state model using hFRET. (D) Dwell time distributions for the individual states of RPA–DBD-A^MB543. The dwell times were binned with a bin size of 2.4 s (bars). Solid lines represent exponential fits for each distribution. (E) A representative fluorescence trajectory for the RPA^E240K mutant labeled with MB543 at the DBD-A (RPA^E240K-DBD-A^MB543; orange) overlaid with an idealized trajectory (black). (F) Dwell time distributions for the individual states of RPA^E240K-DBD-A^MB543. The dwell times for states 2, 3 and 4 were binned with a bin size of 2.4 s (blue, yellow and green bars, respectively), while dwell times for state 1 were binned with a bin size 0.2 s (red bars). Solid lines represent exponential fits for each distribution. (G–J) Transition density plots for the RPA–DBD-A^MB543, RPA^E240K–DBD-A^MB543, RPA-DBD-D^MB543, and RPA^E240K-DBD-D^MB543, respectively. Only transitions after the buffer wash were considered in these plots. (K) Summary of the dwell time analysis for all proteins in this study. The dwell times are shown as time constants from the exponential fitting of the dwell time distributions ± fitting error. The percentage of visitations to each state is shown below the respective dwell times.

Figure 2.

Binding and conformational dynamics of RPA–DBD-A^MB543 and RPA^E240K–DBD-A^MB543 on telomeric G-quadruplex DNA.(A) Experimental setup. Biotinylated partial duplex DNA with 3′-ssDNA overhang containing five TTAGGG repeats was prepared under conditions enforcing formation of the G-quadruplex, and tethered to the TIRFM flow cell. At 30 s, the indicated fluorescently-labeled RPA was flowed in, and at 120 s the unbound protein was removed by flowing in buffer. (B–E) Representative MB543 trajectories and transition density plots (TDPs) for RPA-DBD-A^MB543 (B, C) and RPA^E240K-DBD-A^MB543 (D, E), respectively. TDPs were built only from the events after buffer wash.

Figure 3.

RPA and hnRNPA1 reorganize telomeric G-quadruplex DNA and form ternary complex. (A) Bulk FRET-based analysis of the hnRNPA1 binding to 10 nM telomeric G-quadruplex folded in K⁺ containing buffer (open black circles), and to telomeric G-quadruplex melted by RPA (green squares) or RPA^E240K (orange squares). The binding curves were fitted using quadratic binding equation, and the K_ds are shown with their respective fitting errors. Each complex with their respective FRET values are shown schematically on the right labeled with Cy3 (FRET donor) and Cy5 (FRET acceptor) at the two termini. The free and bound DNA substrates are schematically shown on the right with their respective FRET values. The experiments were carried out in triplicates and the values are plotted as average ± standard deviation for the three independent titrations. Note that the error bars for most measurements were smaller than the symbols’ sizes. Apparent equilibrium dissociation constants are shown with their respective fitting errors. (B) hnRNPA1 binding to 10 nM telomeric G-quadruplex folded in Li⁺ containing buffer (open black circles), and to telomeric G-quadruplex melted by RPA (green squares). (C–J) Equilibrium smFRET distributions for the protein-free h-telG4 labeled with the Cy3 and Cy5 dyes in K⁺ (C) and Li⁺ containing buffer (D), h-telG4 bound by saturating amounts of RPA (E, F), hnRNPA1 (G, H) and RPA and hnRNPA1 together (I, J). Green bars and red asterisks mark FRET species specific to the h-telG4/RPA/hnRNPA1 complexes. For all measurements, FRET values collected from 10 randomly chosen fields of view, binned in 0.02 unit bins and plotted as histograms. Experiments were repeated at least three times. A representative set of distributions is shown.

Figure 4.

RPA and hnRNPA1 form ternary complex on telomeric ssDNA. (A–J) The MP analyses of the 50 nM RPA (green), 50 nM RPA^E240K (orange) and 300 nM hnRNPA1 (grey), and their complexes with 50 nM telomeric ssDNA (five TTAGGG repeats) and each other in the Li⁺ containing buffer. Recorded mass values were binned in 3 kDa bins, plotted and fitted with one, two or three Gaussians (see Supplementary Table S2 for details). The optimal number of Gaussians used in each fit was selected based on an F-test. (A–C). Individual proteins. (D–F) Complexes of individual proteins and telomeric DNA. (G, H) Molecular weights of RPA (G) or RPA^E240K (H) mixed with telomeric DNA and hnRNPA1. The new Gaussian peak which we attribute to the ternary RPA–DNA–hnRNPA1 complex is marked with a cartoon representation of the complex. (I, J) Molecular weights of RPA (I) or RPA^E240K (J) mixed with hnRNPA1. (K) hnRNPA1 physically associates with RPA in cell lysates. Cell lysates of PCS201 iPSCs with wild type RPA1 and RPA1 p.E240K were treated with micrococcal nuclease prior to immunoprecipitation with anti-hnRNPA1. N = 3 biologically independent experiments. Histone H3 was used as a loading control.

Figure 5.

hnRNPA1 constrains conformational dynamics of the DBD-A of RPA bound to telomeric ssDNA. (A) Experimental setup of the smTIRFM experiment. (B) Experimental scheme. Biotinylated partial duplex DNA with 3′-ssDNA overhang containing five TTAGGG repeats was prepared in the Li⁺-containing buffer, and tethered to the TIRFM flow cell. At 30 s, the indicated fluorescently labeled RPA was flowed in, and at 120 s the unbound protein was replaced with 50 nM unlabeled hnRNPA1 or protein-free imaging buffer. (C) Representative MB543 trajectories for the wild type RPA-DBD-A^MB543 in the presence (top) or absence (bottom) of hnRNPA1. Asterisks mark deviation of the fluorescence signal from the lowest state suggestive of the presence of RPA. (D) Representative MB543 trajectories for the RPA^E240K–DBD-A^MB543 in the presence (top) or absence (bottom) of hnRNPA1. The signal level corresponding to the RPA^E240K–hnRNPA1-DNA ternary complex is marked by an arrow and a carton representation of the complex.

Figure 6.

RPA-hnRNPA1 complex on telomeric ssDNA is remodeled by TERRA RNA. (A) Experimental scheme. Short movies were recorded under equilibrium conditions in the Li⁺-containing buffer (1) in the presence of tethered DNA, (2) upon addition of 100 pM of indicated fluorescently labeled RPA, (3) upon replacement of unbound RPA with 50 nM hnRNPA1, and (4) upon replacement of hnRNPA1 with buffer or 50 nM (molecules) TERRA RNA. Representative color-inverted images of the 1/4 of the field of view are shown in panels (B), (E), (H) and (K). Number of trajectories with the fluorescence signal above background recorded in five short movies under each conditions are quantified in panels (C), (F), (I) and (L). Fluorescence in each point of these trajectories is summarized in panels (D), (G), (J) and (M). Statistical analysis: ordinary one-way ANOVA (GraphPad Prism).

Figure 7.

Telomere-specific RPA–DNA–hnRNPA1 complex.(A) Several distinct events at human telomeres may lead to formation of G-quadruplexes that require RPA presence: (1) replication of the telomeric DNA and (2) transcription of TERRA RNA expose the G-strand, while (3) the end of human telomeres is a 50–500 nt 3′ ssDNA overhang. (B) RPA forms a long-lived, macroscopically stable but microscopically dynamic complex on telomeric ssDNA. Formation of the RPA–DNA–hnRNPA1 complex constrains RPA dynamics potentially playing a protective role until RPA is replaced at the ssDNA overhangs with telomere-specific POT1/TTP1. (C) Stable RPA-hnRNAPA1 complex with constrained conformational dynamics may present a nucleoprotein structure refractory to DNA damage signaling, while providing access for the POT1/TTP1 without leaving the DNA deprotected.