The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia

Abstract

Paraneoplastic opsoclonus myoclonus ataxia (POMA) is a neurologic disorder thought to be mediated by an autoimmune attack against onconeural disease antigens that are expressed by gynecologic or lung tumors and by neurons. One POMA disease antigen, termed Nova-1, has been identified as a neuron-specific KH-type RNA-binding protein. Nova-1 expression is restricted to specific regions of the central nervous system, primarily the hindbrain and ventral spinal cord, which correlate with the predominantly motor symptoms in POMA. However, POMA antisera recognize antigens that are widely expressed in both caudal and rostral regions of the central nervous system, and some patients develop cognitive symptoms. We have used POMA antisera to clone a cDNA encoding a second POMA disease antigen termed Nova-2. Nova-2 is closely related to Nova-1, and is expressed at high levels in neurons during development and in adulthood, and at lower levels in the adult lung. In the postnatal mouse brain, Nova-2 is expressed in a pattern that is largely reciprocal with Nova-1, including high levels of Nova-2 expression in the neocortex and hippocampus. Functional characterization of Nova-2 in RNA selection and nitrocellulose filter-binding assays reveals that Nova-2 binds RNA with high affinity and with sequence specificity that differs from Nova-1. Our results demonstrate that the immune response in POMA targets a family of highly related sequence-specific neuronal RNA-binding proteins. The expression pattern of the Nova-2 protein is likely to underlie the development of cognitive deficits in some POMA patients.

Figure 1

Identification of Nova-2, a PND antigen. (A) Western blot analysis of POMA antigens in P0 mouse brain. Total protein extracts (50 μg per lane) from P0 mouse forebrain (FB) or hindbrain (HB) were analyzed by Western blot analysis with POMA antiserum. Molecular mass markers are indicated on the left (kDa). (B) The full-length human Nova-2 amino acid sequence is shown compared with Nova-1. The Nova-1 sequence includes mini-exon LSK (aa 88–90) and alternatively spliced Exon H (aa 153–176). KH domains are boxed in gray. A potential NLS is boxed in white. The peptide sequence used to generate the Nova-2-specific antibody N2Ab (aa 392–405) is underlined. Dashes indicate gaps inserted to align the sequences, dots indicate identity, and asterisks indicate predicted stop codons. (C) The N2FP is recognized by 5 of 5 POMA patient antisera. Western blots with recombinant N2FP (500 ng per strip) were immunoblotted with POMA antisera (lanes 5–9, serum from five different POMA patients at 1:200, 1:250, 1:250, 1:1000, 1:2000), non-POMA paraneoplastic antisera (lane 3, Yo patient serum at 1:200; lane 4, Nb patient serum at 1:200), or normal human control sera (lane 1, 1:50; lane 2, 1:200). Molecular mass markers are indicated on the left (kDa). (D) The antipeptide antibody N2Ab immunoprecipitates a subset of Nova proteins from mouse brain. Immunoprecipitations from E17 mouse brain extracts were performed with affinity-purified N2Ab (lane 3) or a control affinity-purified rabbit antibody (lane 2). Immunoprecipitates were analyzed for Nova proteins by immunoblotting with POMA antiserum. Extract (50 μg) was loaded (lane 1) to demonstrate the Nova proteins in the starting material. Molecular mass markers are indicated on the left (kDa); the Nova-2 50-kDa band (arrowhead) and IgG bands (asterisk) are indicated on the right.

Figure 2

Expression analysis of Nova-2 in mouse. (A) Nova tissue Western blot. Total protein (50 μg per lane) from adult mouse tissues was immunoblotted with POMA antiserum. Lanes: 1, brain; 2, kidney; 3, heart; 4, spleen; 5, lung. Molecular mass markers are indicated on the left (kDa). (B) Nova-2 tissue Northern blot. Total RNA (20 μg per lane) was isolated from adult mouse tissues and hybridized on a Northern blot with a Nova-2 specific probe. Lanes: 1, brain; 2, kidney; 3, heart; 4, spleen; 5, lung. Molecular weight markers are indicated on the left (kb). The blot was stripped and reprobed for actin to demonstrate RNA integrity (data not shown). (C and D) Nova-2 transcripts in the mouse embryo and perinatal mouse. Sagittal sections (12 μm) of E14 embryo (C) or P0 (D) mouse were hybridized with a ³³P-radiolabeled Nova-2 riboprobe and imaged with darkfield microscopy (positive signal is white). At E14, Nova-2 mRNA is detected throughout the CNS, including brain (br) and spinal cord (sp). At P0, Nova-2 transcripts are most abundant in the cortex (ctx), with strong signal also in olfactory bulb (ob), thalamus (th), inferior colliculus (ic), and inferior olive (io). Signal is relatively weak in the brainstem (arrowhead). No signal was seen with control sense riboprobes (data not shown).

Figure 3

Comparison of Nova-2 expression with Nova-1 at P0. Reciprocal patterns of Nova-2 and Nova-1 protein expression at P0. Nova-2 immunoreactivity in sagittal sections of a P0 mouse brain was assessed with affinity-purified N2Ab (a, c, e, g, i), and compared in serial sections with POMA antiserum under fixation conditions previously demonstrated to preferentially detect the Nova-1 protein (ref. ; b, d, f, h, j). Whole brain (a, b), and higher magnification views comparing Nova-1 and Nova-2 expression showing the thalamus [th; (c, d)], inferior olive [io; (e, f)], superior colliculus (sc), inferior colliculus (ic), external and internal granule cell layers of the cerebellum (egl/igl), and deep cerebellar nuclei [dcn; (g, h)], and dorsal (d) and ventral (v) spinal cord (i, j). Arrowhead indicates the large motor neurons in the ventral spinal cord (i, j), where moderate Nova-2 and strong Nova-1 expression is overlapping. Olfactory bulb (ob), cortex (ctx), and pontine nuclei/medioventral periolivary nucleus (pn/mvpo) are also shown.

Figure 4

Nova-2 is an RNA-binding protein. (A) Analysis of Nova-2 RNA-binding ability to ribohomopolymer RNA in varying salt concentrations. N2FP (5 μg) or an irrelevant T7 epitope-tagged and histidine-tagged control protein was incubated with poly(G), poly(A), poly(U), and poly(C)-Sepharose ribohomopolymer beads in either 0.1 M, 0.25 M, 0.5 M, or 0.75 M NaCl. After being washed, protein bound to the ribohomopolymers was analyzed by Western blot analysis with an anti-T7 antibody (Novagen). Twenty percent of the input protein is shown (Lower). (B) Affinity elution-based RNA selection identifies a Nova-2 consensus binding sequence. Alignment of 12 Nova-2 RNA ligands selected in two independent Nova-2 selection experiments (in 0.5 and 0.3 M LiCl) is shown. A consensus sequence is shown at the top, with the GAGUCAU motif highlighted. The match to the consensus, scored as the number of exact matches of 12 sequences (n/12), is shown for each position. Bases in RNA ligands with identity to the GAGUCAU motif are also shown highlighted. (C and D). Direct comparisons of Nova-1 and Nova-2 binding to selected RNA ligands. Nitrocellulose filter binding assays were performed to examine Nova-1 (open squares) and Nova-2 (solid circles) binding to RNA ligands mSB2 (C), a 21-nt RNA containing the Nova-1 consensus binding motif [UCAU(N)_0–2]₃ (19); and 87 (D), a 96-nt RNA ligand isolated by Nova-2 RNA selection (Fig. 4B). RNA ligand mSB2 bound to Nova-1 and Nova-2 with calculated K_d values of 33 ± 5 nM and 73 ± 13 nM, respectively (calculated by least squares fit; ref. 33). Similar results were found with the 96 nt SB2 Nova-1 RNA ligand (data not shown). RNA ligand 87 bound to Nova-1 and Nova-2 with calculated K_d values of 13 ± 2.5 nM and 11 ± 1.5 nM, respectively. Maximal Nova-1 binding to RNA ligand 87 was observed at ≈65% of the total RNA, while maximal Nova-2 binding was 100%. Similar results were found with the Nova-2 RNA ligand 29 (data not shown). Neither Nova protein demonstrated significant binding to a random RNA ligand isolated from the original RNA selection pool, and an irrelevant T7 epitope-tagged recombinant control protein did not demonstrate significant binding to any of the RNA ligands (calculated K_d values > 1 μM, data not shown).