Most eukaryotic genes are expressed as precursor messenger RNAs (pre-mRNAs) that are converted to mRNAs by splicing, an essential step of gene expression. Splicing is catalyzed by the spliceosome, a large and dynamic ribonucleoprotein machine, which removes non-coding intervening sequences (introns) from pre-mRNAs and ligates flanking coding sequences (exons) to produce mature mRNAs for protein synthesis in the cytoplasm. In humans, nearly all pre-mRNAs undergo alternative splicing to produce multiple mRNA isoforms with distinct functions, greatly expanding the complexity of the human transcriptome and proteome. Further, alternative splicing is regulated in time and space, playing critical roles in many biological processes. In fact, mutations and misregulation of splicing have been associated with a large number of human diseases. While in vitro splicing can occur in the absence of transcription, in vivo splicing is intimately coupled with transcription both physically and functionally. However, our understanding of co-transcriptional splicing remains incomplete in part due to limited information on the timing of splicing relative to transcription. Splicing is also a highly accurate process. To promote fidelity in splicing, the spliceosome discriminates and discards suboptimal splicing substrates that have engaged the spliceosome. Although nuclear quality control mechanisms have been proposed to retain immature mRNPs, discarded splicing substrates, including lariat intermediates, do export to the cytoplasm. However, the mechanism for exporting these species has remained unknown. Here, we investigated the dynamics and regulations of co-transcriptional splicing in humans and characterized the nuclear export pathway of lariat intermediates in yeast. To study co-transcriptional splicing in humans, we developed a genome-wide approach, CoLa-seq, or co-transcriptional lariat sequencing. We show that CoLa-seq enables efficient mapping of branch points, an essential reactant in the splicing reaction, in a cell type-specific manner. Additionally, for the first time in vivo, we show that adjacent introns can undergo concurrent splicing. Notably, we provide evidence that the timing of splicing can vary dramatically both across introns and even within the same intron. Further, we show that the splicing of a given intron can occur both through intron definition and exon definition pathway, which has implications for the regulation of alternative splicing. Importantly, we identified key cis-elements and trans-acting factors that correlate with the timing of co-transcriptional splicing. Using a combination of ensemble and single molecule approaches, we demonstrate that the spliceosome-discarded lariat intermediates use the same nuclear export pathway as mature mRNAs do. Further, our findings suggest that during mRNA export, Tom1-mediated ubiquitylation of Yra1p undocks mRNA from the nuclear basket of the nuclear pore, allowing mRNA to transit through the nuclear pore for export.