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. 2021 Nov 25;49(21):12284–12305. doi: 10.1093/nar/gkab1120

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — The Hnrnpr gene encodes two hnRNP R protein isoforms generated by alternative splicing. (A) Western blot analysis of hnRNP R isoforms using a C-terminal-specific antibody performed on tissue lysates from P5 wildtype mice. Equal protein loading in individual lanes was verified by probing with an antibody against β-Actin. (B) Western blot analysis of hnRNP R on brain lysates prepared from wildtype mice of different ages using the C-terminal-specific hnRNP R antibody. Equal protein loading in individual lanes was verified by probing with an antibody against Gapdh. (C) Schematic representation of the murine Hnrnpr locus that gives rise to a full-length Hnrnpr-FL mRNA and an exon 2-skipped Hnrnpr-ΔN mRNA produced by alternative splicing. Full‐length hnRNP R-FL and N‐terminally truncated hnRNP R-ΔN isoforms are shown. Protein domains are depicted as colored boxes: RRM (RNA-Recognition Motif), NLS (Nuclear Localization Signal), RGG (Arg-Gly-Gly box), and Q/N (Gln/Asn-rich domain). Constitutive exons are represented by gray boxes. Exon 2 (red box) is alternatively spliced. Introns are represented by horizontal lines. (D) Western blot analysis of lysates from HEK293TN cells transfected with plasmid constructs for expression of HA-tagged hnRNP R-FL and hnRNP R-ΔN. Total lysates were derived from HEK293TN cells 48 h after transfection with the indicated plasmid, and probed with anti-hnRNP R or anti-HA antibodies. GAPDH was probed as loading control. (E, F) Absolute copy numbers of Hnrnpr-FL (E) and Hnrnpr-ΔN (F) mRNA isoforms in tissues from P5 wildtype mice. Exon junction-spanning primers were used to measure isoform expression by qPCR. 7SL was used for normalization. Data are mean ± Standard Deviation (SD) (n = 3 animals). (G) Ratio of Hnrnpr-FL to Hnrnpr-ΔN isoform expression measured by qPCR. Data are mean ± SD (n = 3 animals). Statistical analysis was performed using a one-sample t-test comparing the ratios to a hypothetical mean of 1; *P ≤ 0.05, **P ≤ 0.01. (H) qPCR analysis of Hnrnpr-FL and Hnrnpr-ΔN isoform expression in brains of wildtype mice of different ages. The Gapdh transcript was used for normalization. Data are mean ± SD (n = 3 animals). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P ≤ 0.01.