# Unraveling the Diversity of RANBP2: Protein Isoforms and Implications for Cellular Function and Human Disease

Sophie Desgraupes, Nathalie Arhel

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Sophie Desgraupes, Nathalie Arhel. Unraveling the Diversity of RANBP2: Protein Isoforms and Implications for Cellular Function and Human Disease. Journal of Molecular Biology, 2025, 437 (24), pp.169452. 10.1016/j.jmb.2025.169452. hal-05398585

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# Unraveling the Diversity of RANBP2: Protein Isoforms and Implications for Cellular Function and Human Disease

Sophie Desgraupes, and Nathalie Arhel

Institut de Recherche en Infectiologie de Montpellier (IRIM), University of Montpellier, France

Correspondence to : sophie.desgraupes@irim.cnrs.fr (S. Desgraupes), nathalie.arhel@cnrs.fr (N. Arhel), https://doi.org/10.1016/j.jmb.2025.169452

Edited by Eric O. Freed

## Abstract

RANBP2 (also known as Nup358) is the largest nucleoporin of the nuclear pore complex (NPC), where it constitutes the main component of the cytoplasmic filaments and regulates nucleocytoplasmic transport. In addition to its NPC-associated functions, RANBP2 also localizes in the endoplasmic reticulum (ER) at annulate lamellae (AL), at mitochondria-ER junctions, and at kinetochores during mitosis, where it contributes to multiple cellular processes including metabolism and mitotic progression. Although most studies focus on the canonical full-length protein (~358 kDa in humans), multiple smaller bands have been detected across species, suggesting the existence of alternative isoforms. Here, we review all predicted and experimentally supported RANBP2 transcript variants, summarize their structural features and discuss their possible origins, including alternative splicing, genomic recombination and proteolysis. We examine how isoform-specific changes, such as loss of the zinc finger domain, Ran-binding domains, E3 SUMO ligase, or the cyclophilin-like domain, could alter RANBP2's cellular functions. We also consider evidence for cell-type specific and developmentally regulated expression from non-human models, and evaluate the potential relevance of RANBP2 isoforms in viral infections and neurological disease. By compiling genomic, proteomic, and functional data, this review highlights the need for isoform-resolved approaches to fully understand RANBP2 biology and its contribution to human disease.

© 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

The nuclear pore complex (NPC) is a large and dynamic structure, approximately 110 MDa in humans, that mediates the transport of molecules between the cytoplasmic and nuclear compartments [1]. While small molecules can diffuse freely through the pores, molecules larger than 40 kDa require active transport involving nucleoporins (Nups), the protein constituents of the NPC. Roughly 30 different Nups assemble in multiple copies to form an NPC, which is organized into three main structures with eight-fold symmetry: the central ring, the nuclear basket and the cytoplasmic fibrils [2]. While each con-

tributes to nucleocytoplasmic transport, the central ring serves as the primary selective barrier. For import of macromolecules, transport factors called karyopherins bind cargoes and sequentially interact with FG-repeat Nups to mediate translocation across the NPC. The cytoplasmic fibrils participate in nucleocytoplasmic transport by providing direction through the recycling of transport factors, and by releasing, retaining or modifying cargo [3,4]. Several Nups localize to the cytoplasmic side of the NPC, including Nup214, Nup88, Nup62, Nup98, Nup42 and Nup358. However, Nup358, also called Ran-binding protein 2 (RANBP2), is the main constituent of the cytoplasmic fibrils [5].
RANBP2 is present as pentamers within each of the eight cytoplasmic fibrils of the NPC [5], but it is also found at other subcellular sites. In addition to its roles at the nuclear pore, human RANBP2 localizes to the endoplasmic reticulum (ER) at annulate lamellae (AL), at mitochondria-ER junctions and at kinetochores during mitosis. While its NPC-related functions are primarily linked to nucleocytoplasmic transport, RANBP2's activities outside the NPC have been associated with metabolism, cell cycle control and cellular homeostasis [6-9]. For instance, at AL, RANBP2, together with other Nups, is thought to be stored as pre-assembled building blocks that contribute to the assembly of new NPCs after mitosis [8,10]. It also promotes sister-chromatid separation during mitosis by binding to and SUMOylating the Topoisomerase II α [11], and has been reported to regulate mitochondria-ER contact sites [6]. Other cell-type-specific localizations and functions have been reported in other mammalian species, such as in mouse [12,13].

The breadth of RANBP2's subcellular localizations and functions raises the possibility that it may exist as distinct isoforms. Indeed, smaller RANBP2 bands have been reported by Western blotting in rat cells [14,15], *Drosophila* [16], *Xenopus* [17] and human cells [14,18,19,20]. However, these smaller bands were often weak and inconsistently detected, and thus often dismissed as proteolytic degrada-

tion products of RANBP2 or cross-reactive proteins.

Several RANBP2 isoforms are predicted, but how they contribute to RANBP2's functions, where they localize in the cell, and which domains are affected has not previously been comprehensively addressed. In this review, we compare all predicted RANBP2 isoforms through sequence alignment of RefSeq protein-coding sequences (NCBI), describe the molecular events that may generate these variants, and discuss their potential functions and relevance to disease.

## Human RANBP2 transcript variants

The *RANBP2* gene is located on chromosome 2q13, spans 1,122,820 nucleotides (nt), and is predicted to encode multiple splice variants and protein isoforms. In this review, we focus on the RefSeq protein-coding variants available in the NCBI database (Gene ID 5903). A total of 20 different transcripts have been annotated, ranging in length from 2,896 to 11,810 nt and encoding proteins of 874-3,224 amino acids (aa) (Table 1). Among these, four isoforms (isoforms 1-4) are experimentally confirmed, while the remaining sixteen (representing 8 different transcript variants with two accession numbers each) have been predicted computationally using NCBI's gene prediction algorithms.

<table><caption>Table 1 RANBP2 transcript variants.</caption><thead><tr><th colspan="5">NCBI</th></tr><tr><th>Isoform</th><th>Transcript accession number</th><th>Length (nt)</th><th>Protein accession number</th><th>Length (aa)</th></tr></thead><tbody><tr><td>1</td><td>NM_006267.5</td><td>11,708</td><td>NP_006258.3</td><td>3,224</td></tr><tr><td>2</td><td>NM_001415871.1</td><td>11,786</td><td>NP_001402800.1</td><td>3,250</td></tr><tr><td>3</td><td>NM_001415872.1</td><td>11,705</td><td>NP_001402801.1</td><td>3,223</td></tr><tr><td>4</td><td>NM_001415873.1</td><td>11,732</td><td>NP_001402802.1</td><td>3,232</td></tr><tr><td>X1</td><td>XM_005264002.4<br>XM_054343258.1</td><td>11,810</td><td>XP_005264059.1<br>XP_054199233.1</td><td>3,258</td></tr><tr><td>X2</td><td>XM_011511575.3<br>XM_054343259.1</td><td>11,807</td><td>XP_011509877.1<br>XP_054199234.1</td><td>3,257</td></tr><tr><td>X3</td><td>XM_011511576.4<br>XM_054343260.1</td><td>11,633</td><td>XP_011509878.1<br>XP_054199235.1</td><td>3,199</td></tr><tr><td>X4</td><td>XM_047445367.1<br>XM_054343261.1</td><td>9,393<br>9,381</td><td>XP_047301323.1<br>XP_054199236.1</td><td>2,807</td></tr><tr><td>X5</td><td>XM_005264007.4<br>XM_054343262.1</td><td>8,882</td><td>XP_005264064.1<br>XP_054199237.1</td><td>2,282</td></tr><tr><td>X6</td><td>XM_011511578.3<br>XM_054343263.1</td><td>8,879</td><td>XP_011509880.1<br>XP_054199238.1</td><td>2,281</td></tr><tr><td>X7</td><td>XM_017004624.3<br>XM_054343264.1</td><td>7,879</td><td>XP_016860113.1<br>XP_054199239.1</td><td>874</td></tr><tr><td>X8</td><td>XM_017004625.2<br>XM_054343265.1</td><td>2,896</td><td>XP_016860114.1<br>XP_054199240.1</td><td>904</td></tr></tbody></table>

RefSeq protein-coding variants of RANBP2 are listed. The NCBI database includes twenty different entries, of which four represent confirmed isoforms (isoforms 1-4). The remaining predicted isoforms (Xn) have two accession numbers each, depending on whether they were assembled by the Genome Reference Consortium or the Telomere-to-Telomere Consortium. Transcript lengths are indicated in nucleotides (nt), and for each isoform, the corresponding protein accession number and predicted length in amino acids (aa) are provided.
For each predicted RANBP2 transcript variant, two accession numbers are provided in the NCBI database, corresponding to the consortium who obtained the sequences. The most recent human reference genome, GRCh38, was released by the Genome Reference Consortium (GRC) in 2013 as a result of the Human Genome Project launched in 1990. Although the GRCh38 genome represented the most complete vertebrate genome at the time, it still contained hundreds of unresolved gaps across chromosomes. To address these limitations, the Telomere-to-Telomere consortium (T2T) was established and characterized the missing 8% of the genome, including all centromeric regions and the entire short arms of five human chromosomes [21]. A comprehensive overview of all RANBP2 transcript variants and their corresponding NCBI accession numbers is provided in Table 1.

The *RANBP2* gene comprises 29 exons and encodes a full-length protein of 3,224 aa. While most exons vary in length between 68 and 335 nt, exon 20 is exceptionally large, spanning 5,152 nt and alone encoding 53% of the protein (aa 899–2,616).

The *RANBP2* RNA isoforms (Figure 1) contain a total of 19–29 exons, and share a common sequence in the first 18 exons, except for a 3-nt deletion (AGA) at cDNA position 70 spanning the exon 1–2 junction in isoforms 3, X2 and X6. All other sequence differences are confined to exons 19–24. Among the twenty predicted *RANBP2* transcripts, seven distinct patterns of sequence variation can be identified (Figure 2). These reflect possible abnormal splicing events resulting from the introduction of novel splice-sites, abrupt truncation resulting from the introduction of premature STOP codons, or possibly DNA-level recombination, which has been documented in this genomic region [22–25].

The most frequent pattern, found in the majority of isoforms (X1–X6 and isoform 2), is an insertion between exons 20 and 21, at protein position 2,616, apparently resulting from the retention of 78 nt of the intervening intron (Figure 2a). The second most common alteration, observed in isoforms X1–X3, X5–X6 and isoform 4, is an apparent retention of 24 nt from the intron immediately upstream of exon 24 (Figure 2b). A third common pattern, present in isoforms X5 and

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Structure of the *RANBP2* gene and transcript variants. The *RANBP2* gene (NCBI gene ID: 5903) is composed of 29 exons and is predicted to encode multiple RNA isoforms. Transcript variants are either confirmed (“isoform n”) or predicted (“Xn”). Structural modifications include deletions of exonic sequences (dotted red lines and arrows) (isoform 3, X2–X8), insertions of intronic sequences (solid red lines) (isoform 2, isoform 4, X1–X6 and X8) and the introduction of a premature STOP codon (cyan lines) (X4, X7 and X8).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Molecular events generating RANBP2 alternative transcripts. RANBP2 RNAs arise from different putative molecular events. (a) Isoforms 2, X1–X6 retain a 78-nt-long sequence from intron 20. (b) Isoforms 4, X1–X3, X5 and X6, retain the last 24 nt from intron 23. (c) Isoforms X5–X6 lose the first 2,930 nt of exon 20. (d) Isoform X3 has a deletion in exon 20 due to a 116-nt repeat. (e) Isoform X7 skips exon 19, and its truncation is induced by a STOP codon. (f) Isoform X8 retains 113 nt of intron 18, creating a STOP codon. (g) Isoform X4 inserts exon 21 after exon 23, with small deletions causing a STOP codon.</figcaption></figure>

X6, is a large deletion of the first 2,930 nt of exon 20 (Figure 2c).

The remaining four molecular events are each specific to a single isoform. Isoform X3 carries a 177-nt deletion in exon 20 (positions 4,922–5,099), which may be explained by the presence of a repeated sequence (TGTCAGAATCCAGGT AAACAAAATCAAACTACTTCTGCAGTTTCAACA CCTGCCTCTTCAG) (Figure 2d). Isoform X7 lacks the 95-nt exon 19, consistent with exon skipping, and is also truncated within exon 20 due to the appearance of a premature STOP codon (Figure 2e). Isoform X8 contains an intronic retention of 113 nt after exon 18, which introduces a premature STOP codon (Figure 2f). Finally, isoform X4 is a truncated variant in which part of exon 21 has been introduced immediately after exon 23, and with several small deletions that generate a premature STOP codon (Figure 2g).

## Human RANBP2 protein domains and predicted isoforms

The RANBP2 protein consists of multiple functional domains, including an N-terminal domain (NTD; residues 1–752), four Ran-binding domains (RBD; residues 1,171–1,306, 2,011–2,148, 2,309–2,443 and 2,911–3,045), eight zinc finger domains (residues 1,343–1,809), an E3 SUMO-ligase domain (residues 2,631–2,771) and a cyclophilin-like domain (CLD; residues 3,062–3,224) [26]. The alignment of exon positions with the corresponding protein positions and domains is presented in Figure 3.

The NTD of RANBP2 is 900 residues-long and contains tetratricopeptide repeats (TPR), a leucine-rich domain (LRR) and a region that mediates the oligomerization of RANBP2 into filaments at the NPC. It is responsible for targeting RANBP2 to the NPC, where it anchors into the cytoplasmic ring [5]. In mouse, the LRR is thought to mediate localization to mitochondria and interaction with mitochondrial enzymes that control energy homeostasis [12].

The RBD domains of RANBP2 mediate interaction with the Ran GTPase, therefore participating in the transport of molecular cargoes across the nuclear pores [27].

The zinc finger motifs in RANBP2 are organized into tandem protrusions that contact target molecules. Originally identified as DNA-binding sequences [28], zinc fingers are now known to bind to DNA, RNA, proteins and lipids, depending on their amino acid composition [29–31]. These motifs enable RANBP2 to interact with nucleic acids [32].

The E3 SUMO-ligase domain, is a 33 kDa domain, first described in 2002, that confers SUMO1 ligase activity to RANBP2 [33]. It directly interacts with the E2 enzyme Ubc9 and enables the transfer of SUMO1 to the target protein.

Finally, the C-terminal domain (CTD) of RANBP2 adopts a canonical cyclophilin-like fold and is implicated in protein–protein interactions [34]. It also interacts with microtubule motors via the adaptor protein BICD2 and contributes to centrosome and nuclear positioning during mitosis [35]. In mouse, the CTD of RANBP2 is thought to modulate the ubiquitin–proteasome system [36], while binding to microtubules is mediated by a kinesin-
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Alignment of exon and protein positions in human RANBP2. The coding sequence of RANBP2 is composed of 29 exons spanning positions 1–9,675 nt. Exon boundaries are shown in blue, while the corresponding aa positions are indicated in black. The RANBP2 protein contains several domains: an N-terminal domain (NTD), four Ran-binding domains (RBD), eight zinc finger domains (orange), an E3 SUMO-ligase domain and a cyclophilin-like domain (CLD). Domain positions are indicated in gray.</figcaption></figure>

binding domain (KBD) present between RBD2 and RBD3 [37].

Using Clustal Omega for multiple sequence alignment, we compared all the described RANBP2 isoforms to determine which domains are impacted. For each previously described transcript variant, we detail its impact on the protein isoforms (Figure 4). For every isoform, the affected domains, protein differences and corresponding amino acid positions are presented as relative to the reference isoform (isoform 1).

Among the predicted differences, two insertions are shared by many isoforms. A 26 aa insertion (GFNFSLFKSNPMAFWTSTPSSQPESK) at position 2,616 is present in isoforms 2, and X1–X6. This insertion, which falls outside any annotated functional domain, results from the retention of the intronic sequence located between exons 20 and 21 (Figure 2a). Another common insertion consists of 8 aa (QYFVTSSL) at position 2,763 in isoforms 4, X1–X3, X5–X6, resulting from the retention of a 24 nt-long intronic sequence immediately upstream the exon 24 (Figure 2b).

A deletion affecting isoform X3 spans positions 1,640–1,700, removing a 59 residue-long sequence within the zinc finger domain. This deletion is due to the repeated sequences present within exon 20 (Figure 2d). A larger deletion of 976 residues is observed in isoforms X5 and X6, spanning positions 900–1,876, resulting in the loss of the RBD1 and the zinc finger domain. This deletion is due to the partial loss of exon 20 (Figure 2c).

Several isoforms (X4, X7 and X8) are truncated at the C-terminal end, generating shorter proteins of 2,807, 874 and 904 aa, respectively. Isoform X4 lacks all residues beyond position 2,775, including RBD4 and the CLD. Additionally, its C-terminal sequence is modified due to the insertion of the first 36 nt of exon 21 after exon 23 (Figure 2g),

followed by an 18 base-long sequence ending in a STOP codon (ATAGTCTCGGTCTCCTGA). Isoform X7 is the shortest at 874 aa, retaining the NTD (residues 1–752) and ending with a STOP codon (GGCCCAGTCTATGGCATGAATAG), as a result of exon 19 skipping and truncation of exon 20 (Figure 2e). Isoform X8, similarly short at 904 aa, also lacks most functional domains due to retention of sequences from intron 18, ending with a STOP codon (...GAGAGCCAAGATCTCCTGA) (Figure 2f). Finally, three isoforms exhibit an additional single residue deletion involving the lysine at position 25 (isoform 3, X2 and X6).

## Evidence for the expression of RANBP2 isoforms in human cells

Definitive evidence for the translation of RANBP2 transcript variants would require the detection of splice junction peptides in proteomics datasets. However, splice variants are usually less abundant, which reduces peptide coverage and lowers the probability of detecting splice-exon junctions.

Still, several studies using Western blotting have reported multiple smaller bands for RANBP2 in human cells, predominantly above the 100 kDa marker and most abundantly above 250 kDa, consistent with the predicted molecular weights of the RANBP2 isoforms (Figure 4) [14,18,19,20,38,39,40,41]. Most of these bands disappear upon knockdown of RANBP2, suggesting they are specific.

In contrast, other studies report a single 358 kDa band for human RANBP2 [42,43], suggesting that the detection of additional bands may depend on the antibody used. For example, the PA1-082 antibody (Pierce) targets an epitope shared by all isoforms (aa 1–18), whereas the ab64276 antibody (Abcam) does not recognize isoforms X5–X8 as
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Domain organization of predicted human RANBP2 protein isoforms. The domains and motifs are shown with their aa positions (bold black): N-terminal domain (NTD, aa 1–752, Ran-binding domains (RBD, aa 1,171–1,306, 2,011–2,148, 2,309–2,443, 2,911–3,045), zinc finger domains (orange) (aa 1,343–1,809), E3 SUMO-ligase domain (2,631–2,771) and cyclophilin-like domain (CLD, aa 3,062–3,224). For each isoform, domain organization and sequence differences relative to the reference isoform (isoform 1) are indicated in bold gray. Common patterns include a 26-aa insertion at position 2,616 (isoforms 2, X1–X6) and an 8-aa insertion at position 2,763 (isoforms 4, X1–X3, X5–X6). Isoforms 3, X2 and X6 carry a deleted lysine (K) at position 25; isoforms X5 and X6 share a 976-aa deletion spanning positions 900–1,876; and isoforms X4, X7 and X8 are truncated at positions 2,775, 874 and 904, respectively. Isoform length is indicated on the right. Percentage identity (shown in brackets) was determined using Needleman-Wunsch pairwise alignment against the reference isoform 1. Molecular weights (shown underlined) were predicted from the aa sequences.</figcaption></figure>
they lack the targeted epitope (aa 1,550–1,650). Similarly, antibodies from Proteintech and Affinity Biosciences detect only full-length RANBP2 (>350 kDa), while antibodies from Novus Biologics, Thermo Fisher (Invitrogen, MA1-847), and Genetex report additional bands at ~150 kDa, ~120 kDa and as low as 70 kDa in lysates from human cancer cell lines such as HeLa or HEK-293T.

As a word of caution, some RANBP2 antibodies are predicted to detect RGPD (RANBP2 and GCC2 Protein Domains) proteins since their sequence contains duplicated sequences from RANBP2 (exons 1–21, lacking parts of exons 20–21, fused to the 3'-terminal exons of GCC2) [23]. Likewise, cross-reactivity of RANBP2 antibodies with unrelated proteins has also been reported [13].

## Possible origin of RANBP2 isoforms in human cells

Although there is experimental evidence supporting the expression of bona fide RANBP2 protein isoforms in human cells, it remains unclear whether these arise from alternative splicing, DNA recombination, or post-translational proteolysis.

Some of the isoforms appear to involve the entire or partial removal of exons, consistent with alternative splicing. Moreover, tissue-specific splicing of RANBP2 has been reported, particularly in brain and muscle, where certain variants are associated with differentiation and may be relevant to human disease [44]. Additionally, it has been reported that splicing variability is more frequent in genes encoding nuclear envelope proteins, likely because they contain an above average number of exons [44].

Other studies suggest that RANBP2 isoforms originate from distinct genomic loci as a result of DNA recombination. The human RANBP2 locus on chromosome 2 has undergone extensive recombination and duplication, giving rise to RGPD genes [23,25]. Additional genomic events include the distal duplication of the 5' region of RANBP2, which produced RANBP2L1/RANBP2α [45] and oncogenic fusions with the fibroblast growth factor receptor 1 gene (FGFR1) [46], and anaplastic lymphoma kinase (ALK) [47,48]. Supporting the genomic origin of isoforms, CRISPR-mediated insertion of GFP 5' of the RANBP2 locus results in the detection of a single full-length band with a GFP antibody [49,50].

Regarding proteolytic processing, the full-length 358 kDa RANBP2 isoform has been reported to undergo proteasome-dependent degradation under certain stress conditions, such as viral infection [16]. However, there is no evidence of routine endogenous proteolytic regulation, and smaller RANBP2 bands are not detected upon overexpression of tagged cDNAs encoding RANBP2.

## Possible functional significance of RANBP2 isoforms

Protein isoforms can have distinct subcellular localizations [51–56], restricted expression patterns in specific cell types or tissues [57–60], and dynamic regulation during differentiation [61]. For RANBP2, isoform diversity could enable specialized functions in nucleocytoplasmic transport, cytoskeletal interactions, metabolic regulation, and neuronal signaling.

Based on their predicted domain structure (Figure 4), several isoforms are likely to have markedly divergent functions if expressed. For example, isoforms X7 and X8 lack the E3 SUMO ligase domain, which not only SUMOylates key cellular proteins, including sp100, the GTPase Ran, borealin, topoisomerase II, the antiviral protein TRIM5α, and Argonaute proteins, but also anchors RanGAP1 to the cytoplasmic face of the NPC, which is essential for RanGTP hydrolysis [11,39,40,62,63,64,65]. Isoforms X4–X8, which lack one or several Ran-binding domains, are also predicted to exhibit altered nuclear transport functions. Isoforms X3 and X5–X8 lack part or all of the zinc finger domain, which bears the RNA-binding property of RANBP2.

Isoform X4 additionally lacks the cyclophilin-homology domain, which contains peptidyl-prolyl isomerase activity and has been shown to regulate lentiviral infection and cross-species viral adaptation by mediating capsid docking to NPCs [66–70]. This domain also has chaperone activity, and its absence could significantly impact protein folding and stability [71,72].

It remains unclear whether human RANBP2 isoforms display cell type-specific expression patterns or dynamic regulation during differentiation. In rat primary neurons, shorter RANBP2 isoforms of ~200–250 kDa have been reported and proposed to contribute to neuronal polarization [14]. In mouse, at least two isoforms appear to be differentially expressed during neuronal development: a shorter isoform (~230 kDa) that predominates at early developmental stages, and the full-length protein (~350 kDa), which is enriched in mature neurons [73]. However, subsequent work cautioned that the smaller band is likely the cross-reactive detection of neurofascin [13].

Whether human RANBP2 isoforms differ in their subcellular localization is also unknown. All predicted isoforms retain the NTD, which mediates attachment to the NPC [5,74], suggesting a common capacity for nuclear envelope association. Nonetheless, distinct subcellular localization patterns of RANBP2 have been reported in the same cell type depending on the culture conditions or the antibody used. In HeLa cells, for instance, RANBP2 staining is typically perinuclear, but nuclear distribution and/or cytoplasmic aggregates have also been observed to varying degrees
[66,75,76,77–79]. This raises the possibility that isoforms may differ in their distribution, residency time, stability, or that they may compete for NPC association. Supporting this notion, mouse studies reported that the nuclear rim-associated full-length isoform appears shorter-lived than intranuclear or cytoplasmic forms of RANBP2 [73,80].

Further work will be required to determine whether RANBP2 isoforms confer distinct biological functions or merely represent byproducts of alternative splicing, recombination, or proteolysis. Comparative analyses using multiple isoform-specific antibodies may provide a useful strategy to clarify isoform expression across different cell types and tissues.

## Relevance of isoforms in human disease

RANBP2 participates in many cellular processes, including nucleocytoplasmic transport, mRNA export and remodeling, protein SUMOylation, mitotic progression, chromosome segregation and tumorigenesis. Accordingly, changes in its expression levels, subcellular localization or mutations in its coding sequence are associated with human disease [26].

Isoform diversity is a feature of most human genes, and while isoform abundance and localization are regulated throughout differentiation and development, their imbalance can lead to disease [81,82]. However, it is not currently known whether RANBP2 isoforms might constitute disease variants, nor whether shifts in their relative expression might drive disease.

Viral infection profoundly alters the distribution of RANBP2, by triggering its relocalization away from the NPC to the cytoplasm or to viral factories [83,84], and promoting its degradation [16]. If full-

length RANBP2 is preferentially enriched at the nuclear rim, this redistribution could favor the accumulation of shorter isoforms away from the pore. Depletion of RANBP2 from the NPC is likely to have major structural consequences since RANBP2 stabilizes the cytoplasmic ring [85] and its loss results in NPCs lacking both cytoplasmic filaments and ring [17,85].

RANBP2 is also a genetic risk factor for Acute Necrotizing Encephalopathy type 1 (ANE1), a severe neurological disorder characterized by recurrent episodes of acute neurological damage. Several autosomal dominant point mutations in RANBP2 have been linked to ANE1, the most frequent being c.1754C>T (p.T585M) [86,87] (Figure 5). Some of these mutations occur in a region that is shared across all variants (c.128A>T, c.872A>G, c.1350A>T, c.1754C>T, c.1958C>T, c.1966A>G and c.2043G>C), while others map to exon 20 and are absent from some isoforms. For instance, mutations c.3363G>T, c.4993A>G and c.5249C>G are absent from the X5–X8 isoforms, and c.4993A>G is also absent from isoform X3. Likewise, mutations c.7454G>T, c.7474 A>G and c.7807C>T are absent from the two shortest isoforms, X7 and X8 (Figure 5).

The most frequent mutation, c.1754C>T/p. T585M, is located in a donor splicing site at the end of exon 12, but it has not been addressed whether this mutation affects splicing. Because it is located in the NTD, all RANBP2 isoforms could in principle carry the mutation. Alternatively, only a subset of variants might express the mutation and exert a dominant negative effect on wild-type variants. A similar pattern of expression has been described for the RGPD gene family, which arose through intra-chromosomal duplication of RANBP2 [23,25]. Of the seven RGPD paralogs in the human genome, four carry the c.1754C>T allele (which

<figure><img src="image_7.png" /><figcaption>**Figure 5.** Positions of ANE1-mutations on the coding sequence of RANBP2. Among the reported ANE1-associated mutations, seven are located in a region that is present on all isoforms (c.128A>T, c.872A>G, c.1350A>T, c.1754C>T, c.1958C>T, c.1966A>G and c.2043G>C). Three mutations are absent from isoforms X5–X8 (c.3363G>T, c.4993A>G and c.5249C>G) and three are absent from the short X7 and X8 isoforms (c.7454G>T, c.7474A>G and c.7807C>T). *: the c.4993A>G mutation is also absent from the X3 isoform, additionally to being absent from isoforms X5–X8. The mutations indicated in black might be generated by recombination with RGPD sequences. The underlined mutation is localized in a donor splicing site.</figcaption></figure>
predisposes to ANE1 in RANBP2), while the other three remain wild-type [88].

Interestingly, isoform imbalances are especially pronounced during neurogenesis, and several neurological diseases, including amyotrophic lateral sclerosis (ALS), autism spectrum disorder (ASD), and schizophrenia, have been linked to isoform dysregulation [89,90]. Despite ANE1 being a neurological disease, RANBP2 expression dynamics in the brain remain uncharacterized, largely due to the lack of a relevant animal model. Mapping RANBP2 isoform expression in mouse brains would provide valuable insight into potential region-specific expression and the impact of ANE1 mutations on isoform abundance or subcellular localization at the tissue level.

Further work is required to determine if RANBP2 variants carry isoform-specific functions and how the absence of one isoform might impact the others. Future efforts should focus on experimentally validating predicted RANBP2 isoforms and characterizing their cell-type specific expression. Also, single-isoform studies will be necessary to define their distinct functions and subcellular distributions. Finally, organ-specific localization in animal models will be crucial to understand how isoform-specific regulation contributes to tissue homeostasis and to the clinical manifestations of RANBP2-related disease.

## CRediT authorship contribution statement

**Sophie Desgraupes:** Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Conceptualization. **Nathalie Arhel:** Writing – review & editing, Supervision, Funding acquisition.

## DATA AVAILABILITY

No data was used for the research described in the article.

## DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Received 4 July 2025;
Accepted 16 September 2025;
Available online 19 September 2025

**Keywords:** RANBP2/Nup358; nuclear pore complex; transcript variants; protein isoforms; nucleocytoplasmic transport

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