U2AF2 controls alternative splicing in speckles proximal regions in an RS domain-dependent manner

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 its RS domain alters numerous of these interactions, reduces its localisation in speckles and impacts splicing genome wide in a manner that depends both on splicing signals and on introns length. Indeed cassette exon flanked by short introns in genes or transcripts located closed to speckles are the most affected by U2AF2 knockdown or RS domain removal. Interestingly, our bioinformatic analyses further reveal that alternative splicing in general mainly occurs near speckles. Finally, we show that multiple phosphorylation sites within the U2AF2 RS domain are required for normal splicing, suggesting that its RS domain mediates U2AF2 regulations. Altogether our data indicate that U2AF2 is in limiting amount to ensure cassette exon inclusion near speckles and give a novel insight on the mechanism of alternative splicing.