Posttranslational modification of the spliceosome paralogue SNRPN links metabolic signaling to splicing regulation
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Abstract
Gene duplication events give rise to paralogues that can evolve specialized functions over time. SNRPB and SNRPN are two such splicing paralogues, sharing over 90% sequence identity yet exhibiting distinct expression patterns, regulatory features, and disease associations. While SNRPB is ubiquitously expressed and essential in most tissues, SNRPN is expressed primarily in the brain and heart and is subject to genomic imprinting. The aim of this work was to investigate how these paralogues differ at the protein level, particularly focusing on their unstructured regions and their responses to cellular context.
To dissect intrinsic differences between the two proteins, we used an inducible expression system in HEK cells to express 2xHA-mNEON-tagged SNRPB and SNRPN individually in the absence of endogenous SNRPB. Transcriptomic analysis revealed that SNRPN and SNRPB stabilize largely non-overlapping sets of transcripts. SNRPN preferentially stabilized genes that are involved in metabolic pathways and mitochondrial function, and SNRPN-expressing cells exhibited higher proliferation rates. In contrast, SNRPB regulated transcripts involved in cell signaling and adhesion. Despite their roles in the spliceosome, these paralogues did not significantly affect alternative splicing; instead, they appeared to modulate global splicing efficiency, particularly of long and complex transcripts.
A striking difference emerged in the posttranslational regulation of SNRPN. Upon translation inhibition with cycloheximide (CHX), SNRPN – but not SNRPB – underwent a loss of detection by the SmB/B’/N (12F5) antibody, a change that correlated with decreased Arginine methylation in its proline-rich C-terminal region. Mass spectrometry revealed several SNRPN-specific methylated Arginine residues, and mutational analysis showed that replacing key Arginine residues with Lysine residues abolished this CHX-sensitive phenotype. Notably, Arginine 228 (R228) was consistently present in all mutation combinations that disrupted the phenotype but was never observed as methylated, suggesting it may instead be citrullinated.
Together, these findings uncover a previously unrecognized regulatory axis in which SNRPN integrates translational and metabolic cues to modulate its posttranslational state and stabilize complex transcripts. This translationally sensitive regulation may explain why SNRPN is enriched in energy-demanding tissues such as the brain. This work establishes a framework for understanding how paralogues with nearly identical sequences can diverge through subtle, context-dependent posttranslational modifications to acquire distinct cellular roles.