SUMO Modification of Heterogeneous Nuclear Ribonucleoproteins

Abstract

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...

Sumoylation in axons triggers retrograde transport of the RNA-binding protein La

“…With the long distances that mRNAs and RNA-binding proteins must travel in neurons, sumoylation may provide a means to recycle some proteins to the nucleus by targeting these proteins for retrograde transport. Sumoylation has recently been demonstrated for a few RNAbinding proteins (19)(20)(21)(22). Sumoylation of hnRNP C decreases its nucleic acid binding (21).…”

“…Sumoylation has recently been demonstrated for a few RNAbinding proteins (19)(20)(21)(22). Sumoylation of hnRNP C decreases its nucleic acid binding (21). Sumoylation at K41 could affect La's interaction with target mRNAs, because this residue lies within the La motif, and deletions of the La motif alter La's affinity for target RNAs (3).…”

Regulation and Function of SUMO Modification

“…For example, nuclear sumoylation of Dictyostelium Mek1 is responsible for its movement to the cytoplasm (37), and mutation of lysine 99 of the TEL protein leads to increased levels of this protein in the nucleus, suggesting a possible role for sumoylation in its nuclear export (38). In addition, sumoylation of heterogeneous nuclear ribonucleoproteins M and C has been proposed to function as a regulator of conformational changes that may influence nucleocytoplasmic transport of these protein complexes (39).…”

Cloning and Characterization of DULP, a Novel Ubiquitin-Like Molecule from Human Dendritic Cells

“…Dendritic cells are the key antigen-presenting cells which process the antigen and initiate immune response; we expected that the study of this novel ubiquitin-like molecule could contribute to our knowledge of the mechanism of immune response. Multiple alignment analysis of DULP, ubiquitin and other ubiquitin-like proteins showed that ubiquitin, NEDD8, UCRP and Sentrin-1 (SUMO-1) all had a diglycine motif situated either directly at the carboxyl terminus of the UBL or within the sequence of a UBL precursor, which is required for conjugation formation (15)(16)(17)(18), but the ubiquitin domain in DULP did not possess the highly conserved C-terminus Gly-Gly for attachment to -amino group of substrate protein's Lys residues. Moreover, the sequence analysis also showed three conserved Lys residues within ubiquitin: Lys29, Lys48 and Lys63.…”