Biophysical characterisation of human LincRNA-p21 sense and antisense Alu inverted repeats

Abstract

Human Long Intergenic Noncoding RNA-p21 (LincRNA-p21) is a regulatory noncoding RNA that plays an important role in promoting apoptosis. LincRNA-p21 is also critical in down-regulating many p53 target genes through its interaction with a p53 repressive complex. The interaction between LincRNA-p21 and the repressive complex is likely dependent on the RNA tertiary structure. Previous studies have determined the two-dimensional secondary structures of the sense and antisense human LincRNA-p21 AluSx1 IRs using SHAPE. However, there were no insights into its three-dimensional structure. Therefore, we in vitro transcribed the sense and antisense regions of LincRNA-p21 AluSx1 Inverted Repeats (IRs) and performed analytical ultracentrifugation, size exclusion chromatography, light scattering, and small angle X-ray scattering (SAXS) studies. Based on these studies, we determined low-resolution, three-dimensional structures of sense and antisense LincRNA-p21. By adapting previously known two-dimensional information, we calculated their sense and antisense high-resolution models and determined that they agree with the low-resolution structures determined using SAXS. Thus, our integrated approach provides insights into the structure of LincRNA-p21 Alu IRs. Our study also offers a viable pipeline for combining the secondary structure information with biophysical and computational studies to obtain high-resolution atomistic models for long noncoding RNAs.

Figure 1.

Organisational Flowchart fossr the Purification and Characterisation of Sense and Antisense LincRNA-p21 AluSx1 RNA. The determination of LincRNA three-dimensional, low-resolution structures overlaid by high-resolution, atomistic models was conducted in three phases: RNA preparation and biophysical studies to determine sample homogeneity and sample properties; low-resolution structure determination by SAXS; and high-resolution modelling using SimRNA, with constraints imposed by HYDROPRO. All methods are further described below.

Figure 2.

Purification of Sense and Antisense in vitro Transcribed LincRNA-p21 AluSx1 RNA. (A) depicts the size exclusion chromatogram of the sense and antisense AluSx1 RNA elution profile using the Superdex 200 Increase GL 10/300 column. SEC-MALS and SV-AUC experiments were performed with the fractions highlighted in red (sense) and blue (antisense). (B) shows the 10% urea PAGE gel used to ascertain the sense and antisense LincRNA-p21 RNA purity extracted using 0.5 mL fractions (volumes in red) using an ÄKTA Pure FPLC through a Superdex 200 Increase GL 10/300 SEC column. Fractions collected at 11.0 mL and 11.5 mL for sense and antisense AluSx1 purifications were consolidated and used for SAXS and SV-AUC experiments. A Quick-Load® Purple 100 bp DNA Ladder (NEB, Canada) was used for the 10% urea PAGE gels in lanes 1 and 7 of each gel. (C) dC/ds sedimentation coefficient distributions for sense (Red) and anti-sense (Blue) under 6M urea denaturing conditions. (D) same as (C), except transformed to molar mass distributions assuming a partial specific volume of 0.516 mL/g.

Figure 3.

Molecular Weight Determination of Sense and Antisense LincRNA-p21 AluSx1 RNA using SEC-MALS. (A) Portrays the elution curve from the Superdex 200 Increase GL 10/300 SEC of sense (Red) and antisense (Blue) AluSx1 RNAs. (B) Demonstrates the absolute molecular weight distribution across the elution peak of sense LincRNA-p21 AluSx1 RNA’s elution profile, and light scattering (blue), UV (red), and RI (purple) scattering. (C) Portrays the absolute molecular weight distribution across the elution peak the results fitting of antisense LincRNA-p21 AluSx1 RNA’s elution profile, and light scattering (Blue), UV (Red), and RI (Purple) scattering.

Figure 4.

Small Angle X-Ray Scattering (SAXS) Characterisation of Sense (red) and Antisense (blue) LincRNA-p21 AluSx1 RNA. (A) merged scattering data of sense and antisense AluSx1 RNA depicting the scattering intensity (log I(q)) vs. scattering angle (q = 4πsinθ/λ). (B) Guinier plots allowing for the determination of R_g from the low-angle region data and representing the homogeneity of samples. (C) Dimensionless Kratky plots (I(q)/I(0)*(q*R_g)2 vs. q*R_g) of sense and antisense AluSx1 RNA depicting the elongated, tube-like structures because of the non-Gaussian, levelled-plateau shape of the curve. (D) Normalised pair distance distribution plots for sense and antisense AluSx1 RNA which permits the determination of R_g derived from the SAXS dataset and including each molecule's D_max.

Figure 5.

Low-Resolution Structures of Sense (A, Grey) and Antisense (B, Pale Cyan) LincRNA-p21 AluSx1 Inverted Repeats Determined using SAXS. (A) The averaged DAMAVER SAXS low-resolution structure of sense LincRNA-p21 AluSx1 RNA, taking on an elongated, asymmetrical, and extended structure with maximum length of 185.0 Å. Key features include a left and right Bulge. (B) The averaged DAMAVER SAXS low-resolution structure of antisense LincRNA-p21 AluSx1 RNA, adopting an elongated, asymmetrical, and extended structure with maximum length of 180.7 Å. Key features include a left bulge, central bulge, and a right protrusion. Dimensions are represented by the D_max obtained from the P(r) analysis. Models are rotated along their x-axis by 90º as represented by the inset.

Figure 6.

The SimRNA High-Resolution, High-Fidelity Models of Sense LincRNA-p21 AluSx1 RNA. Figure 6 presents the high-resolution, high-fidelity sense models that have good fitting with their SAXS envelope as demonstrated by low NSD values. (A) represents model 514; (B) represents model 1036; (C) represents model 1476; (D) represents model 1677; and (E) represents model 1794 which exhibit chemically probed secondary structures: left arm (Blue); 5′-junction (Green); three-way junction (Yellow), right arm (Magenta), and the 3′-adenyl tail (Cyan). Terminal nucleotides are displayed as: 5′nt (Red, Sphere Modelled) and 3′nt (Lime Green, Sphere Modelled). Models are rotated along their x-axis by 90º as indicated by the inset. A flexible, single-stranded linker sequence is represented centrally (Orange).

Figure 7.

The SimRNA High-Resolution, High-Fidelity Models of Antisense LincRNA-p21 AluSx1 RNA. Figure 7 presents the high-resolution, high-fidelity sense models that have good fitting with their SAXS envelope as demonstrated by low NSD values. (A) represents model 66; (B) represents model 974; (C) represents model 1013; (D) represents model 1074; and (E) represents model 1417 which exhibit chemically probed secondary structures: left arm (Blue); 5′-junction (Green); three-way junction (Yellow), right arm (Magenta), and the 5′-uridyl tail (Cyan). Terminal nucleotides are displayed as: 5′nt (Red, Sphere Modelled) and 3′nt (Lime Green, Sphere Modelled). Models are rotated along their x-axis by 90º as indicated by the inset. A flexible, single-stranded linker sequence is represented centrally (Orange).

Figure 8.

Superimposed Overlays of Sense SAXS Envelopes with their High-Resolution, High-Fidelity SimRNA Models. Figure 8 represents the combined overlays of the sense LincRNA-p21 AluSx1 RNA envelope with their top high-resolution, SimRNA models: (A) 514; (B) 1036; (C) 1476; (D) 1677; and (E) 1794. Overall, SAXS envelopes indicate a general agreement with computationally generated structures, showing high overlap of the extended molecule with the major secondary structures identified using chemical probing techniques.

Figure 9.

Superimposed Overlays of Antisense SAXS Envelopes with their High-Resolution, High-Fidelity SimRNA Models. Figure 9 represents the combined overlays of the antisense LincRNA-p21 AluSx1 RNA envelope with their top high-resolution, SimRNA models: (A) 66; (B) 974; (C) 1013; (D) 1074; and (E) 1417. Overall, SAXS envelopes indicate a general agreement with computationally generated structures, showing high overlap of the extended molecule with the major secondary structures identified using chemical probing techniques. However, there is a slight overhang present with the right arm (Magenta) which has an area excluded from overlapping with the SAXS envelope.

Figure 10.

Superimposed Overlays of Alu Crystal Fragments with High-Resolution LincRNA-p21 Sense and Antisense Models. Left panels depict the overall structures and their overlays; right panels present the same but magnified view. (A) represents the crystal structure of archaeal (P. horikoshii) Alu RNA (Red, 4UYK) bound to the Signal Recognition Particle (SRP, 9 kDa) protein (Pink) and overlaid with the high-resolution 1036 model of sense LincRNAp-21 AluSx1 (Blue) (103) (see Supplementary Movie S7). (B) presents the crystal structure of Alu RNA derived from B. subtilis (Red, 4WFL), overlaid with the high-resolution 1036 model of sense LincRNA-p21 AluSx1 (Blue) (104) (see Supplementary Movie S8). (C) shows the overlap of the high-resolution LincRNA-p21 AluSx1 1036 model (Blue) with the high-resolution structure of human Alu RNA fragment (Red, 5AOX) bound to the SRP protein (Pink) (105) (see Supplementary Movie S9). (D) presents the high-resolution antisense LincRNA-p21 AluSx1 1013 model (Red) overlaid with the crystal structure of canine 7S RNA (Blue, 4UE5) bound to SRP protein (Green) (106) (see Supplementary Movie S10). Each Alu crystal fragment generally depicts strong alignment and conservation of secondary structure with the sense LincRNA-p21 AluSx1 model. RNAalign models were visualised by UCSF Chimera (107).