<span class="paragraphSection"><div class="boxTitle">Abstract</div>Splicing factor U2AF2 is known to play a pivotal role for 3′ splice site recognition at an early step of spliceosome assembly. Here, using proximity labeling and biochemical confirmations, we extend the repertoire of putative functional partners of U2AF2 mainly for splicing, chromatin modification, transcription, 3′ end processing, and RNA methylation. Removal of the U2AF2 RS domain alters numerous interactions, including self-association, reduces its localization to nuclear speckles, and impacts splicing genome-wide in a manner that depends both on splicing signals and on intron length. Indeed, cassette exon flanked by short introns in genes or transcripts located close to speckles are the most affected by U2AF2 knockdown or RS domain removal. Finally, we show that phosphorylation sites within the U2AF2 RS domain are required for normal splicing, suggesting that its RS domain mediates U2AF2 regulation. Our in-depth bioinformatics analyses reinforce previous observations that alternatively spliced transcripts accumulate in the proximity of speckles. Our results suggest that although U2AF2 is clearly enriched in these regions, its local concentration remains limiting. Consequently, a global reduction in U2AF2 disproportionately affects splicing in the vicinity of nuclear speckles. This provides new insight into how spatial protein availability contributes to the regulation of alternative splicing.</span>