Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3\' UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a \"leaky\" stop codon context, which likely defines the basis of nonsense suppression.

Alternative polyadenylation (APA) produces mRNA isoforms with different 3\' UTR lengths. Previous studies indicated that 3\' end processing and mRNA export are intertwined in gene regulation. Here, we show that mRNA export factors generally facilitate usage of distal cleavage and polyadenylation sites (PASs), leading to long 3\' UTR isoform expression. By focusing on the export receptor NXF1, which exhibits the most potent effect on APA in this study, we reveal several gene features that impact NXF1-dependent APA, including 3\' UTR size, gene size, and AT content. Surprisingly, NXF1 downregulation results in RNA polymerase II (Pol II) accumulation at the 3\' end of genes, correlating with its role in APA regulation. Moreover, NXF1 cooperates with CFI-68 to facilitate nuclear export of long 3\' UTR isoform with UGUA motifs. Together, our work reveals important roles of NXF1 in coordinating transcriptional dynamics, 3\' end processing, and nuclear export of long 3\' UTR transcripts, implicating NXF1 as a nexus of gene regulation.

Post-transcriptional regulation of gene expression can be achieved through the control of mRNA stability, cytoplasmic compartmentalization, 3\' UTR length and translational efficacy. Spermiogenesis, a process through which haploid male germ cells differentiate into spermatozoa, represents an ideal model for studying post-transcriptional regulation in vivo because it involves a large number of transcripts that are physically sequestered in ribonucleoprotein particles (RNPs) and thus subjected to delayed translation. To explore how small RNAs regulate mRNA fate, we conducted RNA-Seq analyses to determine not only the levels of both mRNAs and small noncoding RNAs, but also their cytoplasmic compartmentalization during spermiogenesis.
Among all small noncoding RNAs studied, miRNAs displayed the most dynamic changes in both abundance and subcytoplasmic localization. mRNAs with shorter 3\' UTRs became increasingly enriched in RNPs from pachytene spermatocytes to round spermatids, and the enrichment of shorter 3\' UTR mRNAs in RNPs coincided with newly synthesized miRNAs that target these mRNAs at sites closer to the stop codon. In contrast, the translocation of longer 3\' UTR mRNAs from RNPs to polysomes correlated with the production of new miRNAs that target these mRNAs at sites distal to the stop codon.
miRNAs appear to control cytoplasmic compartmentalization of mRNAs based on 3\' UTR length. Our data suggest that transcripts with longer 3\' UTRs tend to contain distal miRNA binding sites and are thus targeted to polysomes for translation followed by degradation. In contrast, those with shorter 3\' UTRs only possess proximal miRNA binding sites, which, therefore, are targeted into RNPs for enrichment and delayed translation.