Apoptosis-linked changes in the phosphorylation status and subcellular localization of the spliceosomal autoantigen U1-70K

Abstract

Apoptosis consists of highly regulated pathways involving post-translational modifications and cleavage of proteins leading to sequential inactivation of the main cellular processes. Here, we focused on the apoptotic processing of one of the essential components of the mRNA splicing machinery, the U1-70K snRNP protein. We found that at an early stage of apoptosis, before the cleavage of the C-terminal part of the protein by caspase-3, the basal phosphorylation of the Ser140 residue located within the RNA recognition motif, increases very significantly. A caspase-dependent, PP1-mediated dephosphorylation of other serine residues takes place in a subset of U1-70K proteins. The U1-70K protein phosphorylated at Ser140 is clustered in heterogeneous ectopic RNP-derived structures, which are finally extruded in apoptotic bodies. The elaborate processing of the spliceosomal U1-70K protein we identified might play an important role in the regulated breakdown of the mRNA splicing machinery during early apoptosis. In addition, these specific changes in the phosphorylation/dephosphorylation balance and the subcellular localization of the U1-70K protein might explain why the region encompassing the Ser140 residue becomes a central autoantigen during the autoimmune disease systemic lupus erythematosus. Cell Death and Differentiation (2008) In higher eukaryotes, the process of gene expression includes transcription, pre-mRNA splicing/processing and translation in the cytoplasm. The macromolecular machinery involved in pre-mRNA splicing, the spliceosome, consists of five small nuclear ribonucleoparticles (snRNPs) called U1, U2 and U4-6 snRNP, and a large number of additional splicing factors. 1 Splicing factors are supposedly recruited from storage sites, that is interchromatin granules, in the interchromatin space to transcription sites at the periphery of condensed chromatin. The assembly of the spliceosome is initiated by recognition of the 5 0 splice site of pre-mRNA by U1 snRNP and mediated by serine/arginine-rich (SR) proteins. 2 Phosphorylation and dephosphorylation both have an important role in the recruitment of splicing factors and the subsequent regulation of spliceosome assembly and splicing catalysis. 3 The U1-snRNP-specific U1-70K protein is a highly phosphorylated protein, which contains two SR domains. Phosphorylation of both the SR protein ASF/SF2 and the first SR domain of the U1-70K protein are involved in vitro in their interaction. 4,5 In addition, thiophosphorylation of the U1-70K protein, making it resistant to dephosphorylation by phosphatases, results in complete inhibition of splicing, but not of spliceosome formation. 6 At least 11-13 natural variants of the U1-70K protein, partially due to phosphorylation, have been found by 2D analysis. 7,8 However, very little is known about specific phosphorylated residues in this protein or specific enzymes involved in the functional regulation of the U1-70K protein by phosphorylation/dephosphorylation.Apoptosis is a fast and orderly process consisting of a seq...

Mouse dendritic cells matured by ingestion of apoptotic blebs induce T cells to produce interleukin‐17

“…Apoptotic cell bodies also have been termed late apoptotic or secondary necrotic cells. During apoptosis, various modifications of autoantigens (e.g., chromatin) may take place, including cleavage by proteinases, caspases, and/or endonucleases and specific posttranslational modifications (6,(11)(12)(13)(14)(15). These findings suggest that apoptotic blebs are a source of (modified) autoantigens and that they may play a key role in the development of the antichromatin response in SLE.…”

Post-translational modifications, subcellular relocation and release in apoptotic microparticles: apoptosis turns nuclear proteins into autoantigens.

“…HERDS contain many RNA-associated autoantigens, such as the U1-70K protein ( Fig. 1 and ref [20]) and the Sm antigen [54], which are not degraded since they are still detectable by antibody probes. In addition, HERDS contain still detectable RNA [56].…”

“…The apoptotic cell has been proposed as the source of nuclear autoantigens targeted during lupus, since (i) nuclear autoantigens were identified in apoptotic surface blebs [8]; (ii) mice deficient in molecules involved in the removal of apoptotic cells develop lupus-like autoimmunity [9,10]; (iii) apoptotic cells, especially in combination with dendritic cells (DCs) are able to activate autoreactive T cells and induce an anti-nuclear immune response in mice [11][12][13]; and (iv) autoantibodies from lupus patients and mice preferably recognize modified proteins from apoptotic cells [14][15][16][17][18][19][20].…”

“…We have recently described that during apoptosis a specific change in the phosphorylation status of the U1-70K protein occurs (Fig. 1) [20]. First, the phosphorylation of Ser140 in the RNA recognition motif (RRM) increases directly after the induction of apoptosis.…”

Tetramers reveal IL-17–secreting CD4+T cells that are specific for U1-70 in lupus and mixed connective tissue disease

“…This U1-snRNP complex is one component of the spliceosome (1,2). A subset of patients with SLE, and all patients with mixed connective tissue disease (MCTD), develop autoantibodies against U1-snRNP, and U1-70 in particular (1,(3)(4)(5). Anti-snRNP autoantibodies are detectable before overt disease in SLE in what is termed a "pathogenic autoimmunity" phase (6).…”