The design of large genetic circuits requires genetic regulatory devices capable of performing complex logic operations that place no excessive metabolic burden on the host cell. Hybrid riboswitches, synthetically enhanced compact RNA elements (<100 nucleotides) that form a tertiary structure with the ability to specifically bind two different target molecules, can be used to design genetic regulators that emulate Boolean logic. When inserted into the 5' UTR of a messenger RNA, these devices can regulate translation initiation upon specific binding of one or both ligands without the need for additional auxiliary factors. The goal of this study is to design hybrid riboswitches that emulate Boolean NAND logic in yeast. We propose a novel machine learning-based design framework combining high-throughput in vivo screening and deep Bayesian optimization. Through an initial screening, we discovered a hybrid riboswitch with NAND behavior. Using batch Bayesian optimization with an ensemble neural network as surrogate, we improved the NAND functionality of our hybrid riboswitch with respect to a performance score, thereby achieving near-digital NAND behavior. With its focus on model-based and score-driven design, our proposed method can complement experiment-driven approaches by allowing fine grained adaptation of functionality, including constructs sensitive to single nucleotide changes.
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.
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