Small ubiquitin-related modifiers (SUMOs) are proteins that are posttranslationally conjugated to other cellular proteins, particularly those that localize and function in the nucleus. Enzymes regulating SUMO modification localize in part to nuclear pore complexes (NPCs), indicating that modification of some proteins may occur as they are translocated between the nucleus and the cytoplasm. Substrates that are regulated by SUMO modification at NPCs, however, have not been previously identified. Among the most abundant cargos transported through NPCs are the heterogeneous nuclear ribonucleoproteins (hnRNPs). HnRNPs are involved in various aspects of mRNA biogenesis, including regulation of pre-mRNA splicing and nuclear export. Here, we demonstrate that two subsets of hnRNPs, the hnRNP C and M proteins, are substrates for SUMO modification. We demonstrate that the hnRNP C proteins are modified by SUMO at a single lysine residue, K237, and that SUMO modification at this site decreases their binding to nucleic acids. We also show that Nup358, a SUMO E3 ligase associated with the cytoplasmic fibrils of NPCs, enhances the SUMO modification of the hnRNP C and M proteins. Based on our findings, we propose that SUMO modification of the hnRNP C and M proteins may occur at NPCs and facilitate the nucleocytoplasmic transport of mRNAs.The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of highly conserved RNA-binding proteins that have important roles in regulating multiple steps in mRNA biogenesis and function (10,11,23). In the nucleus, the hnRNPs form large complexes with primary RNA polymerase II transcripts that contain more than 20 different hnRNPs ranging in size from 30 to 120 kDa (4, 31). HnRNPs affect mRNA transcription (16, 28), regulate mRNA translation in the cytoplasm (12-14, 18, 21), and are involved in the maintenance of the single-stranded DNA (ssDNA) extensions at chromosome telomeres (7,(12)(13)(14)24). HnRNPs, however, are best known for their roles in regulating the nuclear posttranscriptional events involved in mRNA biogenesis, including regulation of pre-mRNA splicing, pre-mRNA polyadenylation, and 3Ј-end processing (1, 40) and mRNA nuclear export (10).With regard to their role in nuclear export, most hnRNPs remain associated with the mRNAs as they are translocated through nuclear pore complexes (NPCs) and into the cytoplasm (32, 41). Once in the cytoplasm, these hnRNPs are released from the mRNAs by an unknown mechanism and shuttle back into the nucleus. Intriguingly, several hnRNPs, including the hnRNP C proteins, do not shuttle between the nucleus and the cytoplasm and are presumably released from newly formed mRNAs in the nucleus prior to export (32). Exactly where and how the nonshuttling hnRNPs are released from mRNAs in the nucleus is not currently known. Although significant progress has been made in identifying the factors and steps involved in targeting nuclear mRNPs to NPCs for export, very little is still understood about the exact mechanisms involved in their translocat...

SUMO-1 conjugation modulates numerous cellular functions, including the subnuclear localization of its target proteins. The WT1 tumor suppressor encodes a four-zinc finger protein with distinct splicing isoforms. WT1(؊KTS), encoding uninterrupted zinc fingers, functions as a transcription factor and has a diffusely nuclear distribution; WT1(؉KTS), with an insertion of three amino acids (KTS) between zinc fingers three and four, localizes to discrete nuclear speckles, the function of which is unknown. Because the SUMO-1 E2-conjugating enzyme, Ubc9, interacts with WT1, we tested whether sumoylation modulates the cellular localization of WT1. We find here that both WT1 isoforms are directly sumoylated on lysine residues 73 and 177. Although RNA interferencemediated Ubc9 depletion effectively suppresses WT1 nuclear speckles, a SUMO-1-deficient WT1(؉KTS)(K73, 177R) double mutant retains localization to speckles. Thus, direct sumoylation of WT1 is not responsible for its cellular localization, and other sumoylated proteins may target WT1 to these nuclear structures. Identification of other components of WT1-associated speckles is likely to provide clues to their function.