Transcriptionally non-co-linear (NCL) transcripts can originate from trans-splicing (trans-spliced RNA; ‘tsRNA’) or cis-backsplicing (circular RNA; ‘circRNA’). While numerous circRNAs have been detected in various species, tsRNAs remain largely uninvestigated. Here, we utilize integrative transcriptome sequencing of poly(A)- and non-poly(A)-selected RNA-seq data from diverse human cell lines to distinguish between tsRNAs and circRNAs. We identified 24,498 NCL events and found that a considerable proportion (20–35%) of them arise from both tsRNAs and circRNAs, representing extensive alternative trans-splicing and cis-backsplicing in human cells. We show that sequence generalities of exon circularization are also observed in tsRNAs. Recapitulation of NCL RNAs further shows that inverted Alu repeats can simultaneously promote the formation of tsRNAs and circRNAs. However, tsRNAs and circRNAs exhibit quite different, or even opposite, expression patterns, in terms of correlation with the expression of their co-linear counterparts, expression breadth/abundance, transcript stability, and subcellular localization preference. These results indicate that tsRNAs and circRNAs may play different regulatory roles and analysis of NCL events should take the joint effects of different NCL-splicing types and joint effects of multiple NCL events into consideration. This study describes the first transcriptome-wide analysis of trans-splicing and cis-backsplicing, expanding our understanding of the complexity of the human transcriptome.

Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor-DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF-DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.

Background: Snf1/AMPK activity plays a vital role in adaptation to environmental stress. Results: Elevation of Snf1 phosphorylation restores Snf1 protein in a mutant with low Snf1 levels. Conclusion: Snf1 activity is fine-tuned by a dynamic feedback loop between its protein level and phosphorylation status. Significance: Our results reveal a previously unknown regulatory mechanism of Snf1 activity that is crucial for stress response and aging.

Splicing is initiated by a productive interaction between the pre-mRNA and the U1 snRNP, in which a short RNA duplex is established between the 5′ splice site of a pre-mRNA and the 5′ end of the U1 snRNA. A long-standing puzzle has been why the AU dincucleotide at the 5′-end of the U1 snRNA is highly conserved, despite the absence of an apparent role in the formation of the duplex. To explore this conundrum, we varied this AU dinucleotide into all possible permutations and analyzed the resulting molecular consequences. This led to the unexpected findings that the AU dinucleotide dictates the optimal binding of cap-binding complex (CBC) to the 5′ end of the nascent U1 snRNA, which ultimately influences the utilization of U1 snRNP in splicing. Our data also provide a structural interpretation as to why the AU dinucleotide is conserved during evolution.