Sequence- and structure-specific RNA oligonucleotide binding attenuates heterogeneous nuclear ribonucleoprotein A1 dysfunction

Abstract

The RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (A1) regulates RNA metabolism, which is crucial to maintaining cellular homeostasis. A1 dysfunction mechanistically contributes to reduced cell viability and loss, but molecular mechanisms of how A1 dysfunction affects cell viability and loss, and methodologies to attenuate its dysfunction, are lacking. Utilizing in silico molecular modeling and an in vitro optogenetic system, this study examined the consequences of RNA oligonucleotide (RNAO) treatment on attenuating A1 dysfunction and its downstream cellular effects. In silico and thermal shift experiments revealed that binding of RNAOs to the RNA Recognition Motif 1 of A1 is stabilized by sequence- and structure-specific RNAO-A1 interactions. Using optogenetics to model A1 cellular dysfunction, we show that sequence- and structure-specific RNAOs significantly attenuated abnormal cytoplasmic A1 self-association kinetics and A1 cytoplasmic clustering. Downstream of A1 dysfunction, we demonstrate that A1 clustering affects the formation of stress granules, activates cell stress, and inhibits protein translation. With RNAO treatment, we show that stress granule formation is attenuated, cell stress is inhibited, and protein translation is restored. This study provides evidence that sequence- and structure-specific RNAO treatment attenuates A1 dysfunction and its downstream effects, thus allowing for the development of A1-specific therapies that attenuate A1 dysfunction and restore cellular homeostasis.

Keywords: RNA oligonucleotide; RNA-protein interaction; hnRNPA1; optogenetics; stress granules.

FIGURE 1

OptoA1 clustering is attenuated with the addition of non-sequence specific RNAs. (A) Schematic of BL-inducible A1 self-association clustering using the Cry2PHR optogene (blue), A1 protein (yellow) and mCherry fluorescent tag (red). Representative images of OptoA1 blue light (BL) stimulated cells treated with either (B) No RNA treatment, (C) RNA transfection control, (D) 1.0 µg HEK Total RNA or (E) 1.0 µM IRES RNA. (F) Quantification of A1 cluster formation during a 240-min BL stimulation protocol with the addition of either total RNA collected from HEK293T cells (HEK Total RNA 1.0 ug; Purple) or HIV-1 IRES (IRES) RNA 1.0 µM (Turquoise). Results are plotted as a percent maximum to the highest cluster response at 240 min for HEK Total RNA or IRES RNA treatments, resulting in a kinetics curve for association dynamics. No Treatment (Brown) = no treatment with RNA; Transfection Control (Black) = cells only transfected with RNAiMAX. Dashed lines indicate KA_1/2Max. (F ^I ) Tabular results of a two-way ANOVA, with a Bonferroni post-hoc test from the curves illustrated in (F). (F ^II ) Bar graphs and one-way ANOVA, with a Tukey post-hoc test analysis of KA_1/2Max from the curves illustrated in (F). Data shown are mean±S.E.M. for three biological replicates. Scale bars = 10 µm *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; 95% Confidence Interval.

FIGURE 2

Sequence- and structure-specific RNAO binding to the RRM1 region of A1 alters the thermal stability of A1. Illustrations of (A) MBP-A1WT, (B) MBP-RRMs, (C) MBP-PrLD and (D) MBP Only. DSF melting curves of the thermal unfolding of (A ^I ) MBP-A1WT, (B ^I ) MBP-RRMs, (C ^I ) MBP-PrLD and (D ^I ) MBP Only in the presence of MAX, MED, and LOW RNAOs at different concentrations. Data shown are mean±S.E.M. for three biological replicates. Error bars are omitted for graphical clarity. Dashed lines indicate the melting temperature (T_m). (A ^II ), (B ^II ), (C ^II ) and (D ^II ) Bar graphs and one-way ANOVA, with a Tukey post-hoc test analysis of Tm’s from the curves illustrated in (A ^I ), (B ^I ), (C ^I ) and (D ^I ). NT = No Treatment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; 95% Confidence Interval.

FIGURE 3

3D structural models of the RNAO-RRM complexes and the key residues contributing to their binding free energies (A ^I –A ^III ) The structures of the RNAO-A1 RRM complexes describe that the binding of RNAOs with A1 RRMs were mediated through the interactions of the apical loops of the RNAOs with that of the RNPs from RRM1 and the RRM1/2 linker loop (coloured in purple and shown by arrow in (A ^I ). (B) Close-up views of the binding sites of the complexes reveal key aromatic stacking interactions between RNAOs and RRM. MAX RNAO made two aromatic interactions with PHE17-A13-HIS101 and PHE59-G15, which are consistent with the interactions reported in the known oligos-A1 RRM complexes in PDB (B ^I ). The MED (B ^II ) and LOW (B ^III ) RNAOs exhibit only a single aromatic stacking rendered by a guanine and PHE17. (C) Per-residue decomposition analyses identified other key residues in RRM1 that contributed to the binding free energies of the RNAO-RRM complexes. Several residues from RNPs and the RRM1/2 linker loop contribute to the binding free energy of the MAX complex (C ^I ). The binding free energy of MED-RRM complex is driven mostly by residues from RRM1/2 linker loop and fewer residues from RNPs (C ^II ). Apart from the stacking contact with PHE17, the binding free energy of the LOW-RRM complex is dominated by non-specific electrostatic interactions of residues not part of RNPs (C ^III ).

FIGURE 4

OptoA1 clustering is attenuated with the addition of sequence- and structure-specific RNAOs. Representative images of OptoA1 blue light (BL) stimulated cells treated with either (A) 1.0 µM MAX RNAO, (B) 1.0 µM MED RNAO or (C) 1.0 µM LOW RNAO. (D) Quantification of A1 cluster formation during a 240-min BL stimulation protocol with the addition of either MAX RNAO (Green), MED RNAO (Blue) or LOW RNAO (Red). Results are plotted as a percent maximum to the highest cluster response at 240 min for each RNA treatment, resulting in a kinetics curve for association dynamics. No Treatment = no treatment with RNA; Transfection Control = cells only transfected with RNAiMAX. Dashed lines indicate KA_1/2Max. (D ^I ) Tabular results of a two-way ANOVA, with a Bonferroni post-hoc test from the curves illustrated in (D). (D ^II ) Bar graphs and one-way ANOVA, with a Tukey post-hoc test analysis of KA_1/2Max from the curves illustrated in (D). Data shown are mean ± S.E.M. for three biological replicates. Arrows indicate the formation of OptoA1 clusters. Scale bars = 10 µm *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; 95% Confidence Interval.

FIGURE 5

RNAOs alter the characteristics of OptoA1 clusters. (A) Representative images of OptoA1 blue light (BL) stimulated (240 min) cells treated with either 1.0µM MAX RNAO, 1.0 µM MED RNAO or 1.0 µM LOW RNAO. * indicate cells that contain few, large A1 clusters. Ϟ indicate cells that contain abundant, small A1 clusters. Dashed lines outline cellular nuclei. Scale bars = 10 µm. Quantification of (B) BL stimulated OptoA1 average cluster size and (C) BL stimulated OptoA1 average clusters per cell, combined from three biological experiments. All results were analyzed using a one-way ANOVA, with a Tukey post-hoc test. Data shown are mean±S.E.M. for three biological replicates. *p < 0.05; ***p < 0.001; ****p < 0.0001; 95% Confidence Interval.

FIGURE 6

RNAOs affect the characteristics of SG puncta. (A) Representative images of OptoA1 blue light (BL) stimulated (240 min) cells containing SG puncta treated with and without the co-transfection of either 1.0 µM MAX RNAO, 1.0 µM MED RNAO or 1.0 µM LOW RNAO. * indicate cells that contain both few, large A1 clusters, and large SG puncta. Ϟ indicate cells that contain both abundant, small A1 clusters and small SG puncta. Arrows indicate the formation of SG puncta. Scale bars = 10 µm. Quantification of (B) SG puncta size and (C) SG puncta per cell, combined from three biological experiments. All results were analyzed using a one-way ANOVA, with a Tukey post-hoc test. Data shown are mean±S.E.M. for three biological replicates. *p < 0.05; ***p < 0.001; ****p < 0.0001; 95% Confidence Interval.

FIGURE 7

RNAO treatment attenuates OptoA1 cluster induced cell stress and improves cellular translation. OptoA1 transfected HEK293T cells were BL stimulated for 240 min either without RNAO (A) or with RNAO (B) co-transfection, protein was extracted, and Western blots were probed with phospho-eIF2S1, total eIF2S1 and β-actin antibodies. Additionally, the RNAO co-transfected samples were also probed with mCherry and hnRNPA1 antibodies to detect any changes in OptoA1 in the samples (C). (D) Densitometric analysis of Western blots in (A) and (B). Representative images of OptoA1 BL stimulated cells containing p-eIF2S1 (E) or puromycin incorporation (F) treated with and without the co-transfection of 1.0µM MAX RNAO. ^ indicate cells that contain few, large A1 clusters. # indicate cells that contain abundant, small A1 clusters. * indicate cells that contain both few, large A1 clusters, and large SG puncta. Ϟ indicate cells that contain both abundant, small A1 clusters and small SG puncta. Arrows indicate the formation of SG puncta. Scale bars = 10 µm. (G) Quantification of cell stress activation via p-eIF2S1 fluorescence expression at 45, 80 and 240 min of BL stimulation from cells transfected with 1.0 µM of either MAX, MED or LOW RNAO (images analyzed for MED and MIN RNAOs are like those in (E), which are representative of MAX RNAO). (H) Quantification of cellular translation via percent puromycin incorporation from cells transfected with 1.0 µM of either MAX, MED or LOW RNAO (images analyzed for MED and MIN RNAOs are like those in (E), which are representative of MAX RNAO). All results were analyzed using a one-way ANOVA, with a Tukey post-hoc test. Data shown are mean ± S.E.M. for three biological replicates. *p < 0.05; ***p < 0.001; ****p < 0.0001; 95% Confidence Interval.