Nucleic-acid detection via isothermal amplification and collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative polymerase chain reaction (qPCR). Here, we report the clinical validation of the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) -the virus that causes COVID-19 (coronavirus disease 2019) -in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per

The elongation step of RNA polymerase II (RNAPII) transcription is emerging as a critical control point for the expression of various genes and for diverse biological processes. Examples include neuronal fate determination during embryonic development (6, 44), gene expression of human immunodeficiency virus (5,11,13,19,43), replication and transcription of hepatitis delta virus (38), and transcriptional regulation of heat shock genes (1,10,18). In all these cases, the involvement of three transcription elongation factors, namely, DRB (5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF), NELF (negative elongation factor), and positive transcription elongation factor b (P-TEFb), has been demonstrated or implicated.Shortly after the initiation of transcription, RNAPII comes under the negative and positive control of DSIF, NELF, and P-TEFb. DSIF and NELF cause transcriptional pausing through physical association with RNAPII. DSIF binds to RNAPII directly and stably (33, 36). However, this appears to have little effect on the catalytic activity of RNAPII (37). A previous study has pointed out that NELF does not bind substantially to DSIF or RNAPII alone but does bind to the complex of DSIF and RNAPII (40). This association is the likely trigger of transcriptional pausing. Conversely, P-TEFb allows RNAPII to enter the productive elongation phase by preventing the action of DSIF and NELF (27, 37). P-TEFb is the protein kinase whose primary target is thought to be the C-terminal domain (CTD) of RNAPII (26). Most, but not all, evidence suggests that P-TEFb-dependent phosphorylation of the CTD facilitates the release of DSIF and NELF from RNA-PII, thereby reversing the inhibition (3, 24, 37). In theory, such regulation at the elongation step allows for rapid change in mRNA levels and for highly sophisticated control over gene expression when combined with regulation at the (pre)initiation step.The structures and functions of DSIF and P-TEFb have been extensively characterized. Human DSIF is a heterodimer composed of p14 (14 kDa) and p160 (160 kDa), whose Saccharomyces cerevisiae counterparts are Spt4 and Spt5 (7,33). In addition to its role in transcriptional pausing, DSIF has a potential to activate RNAPII elongation. The activation mechanism is not well understood: interaction partners of DSIF other than NELF may be involved (13,14,20,23,28). Spt5 has a highly acidic N-terminal region, multiple copies of the KOW motifs, and a repetitive C-terminal region analogous to the RNAPII CTD (9,25,36). RNAPII interacts with Spt5 through a region encompassing the KOW motifs. KOW motifs are also found in the bacterial transcription elongation factor NusG, which binds to prokaryotic RNA polymerase and controls termination and antitermination (15,17,29). In addition, the extreme C terminus of Spt5 is specifically involved in the transcriptional repression pathway (6). Human P-TEFb is a heterodimer composed of Cdk9 (41 kDa) and one of multiple cyclin subunits T1, T2a, T2b, and K (50 to 90 kDa) (26). The k...

No abstract

The conserved Prp19 complex (Prp19C) - also known as NineTeen Complex (NTC) - functions in several processes of paramount importance for cellular homeostasis. NTC/Prp19C was discovered as a complex that functions in splicing and more specifically during the catalytic activation of the spliceosome. More recent work revealed that NTC/Prp19C plays a role in transcription elongation in Saccharomyces cerevisiae and in genome maintenance in higher eukaryotes. In addition, mouse PRP19 might ubiquity late proteins targeted for degradation and guide them to the proteasome. Furthermore, NTC/Prp19C has been implicated in lipid droplet biogenesis. In the future, the molecular function of NTC/Prp19C in all of these processes needs to be refined or elucidated. Most of NTC/Prp19C's functions have been shown in only one or few organisms. However, since this complex is highly conserved it is likely that it has the same functions across all species. Moreover, one NTC/Prp19C or different subcomplexes could function in the above-mentioned processes. Intriguingly, NTC/Prp19C might link these different processes to ensure an optimal coordination of cellular processes. Thus, many important questions about the functions of this interesting complex remain to be investigated. In this review we discuss the different functions of NTC/Prp19C focusing on the novel and emerging ones as well as open questions.

Different steps in gene expression are intimately linked. In Saccharomyces cerevisiae, the conserved TREX complex couples transcription to nuclear messenger RNA (mRNA) export. However, it is unknown how TREX is recruited to actively transcribed genes. Here, we show that the Prp19 splicing complex functions in transcription elongation. The Prp19 complex is recruited to transcribed genes, interacts with RNA polymerase II (RNAPII) and TREX, and is absolutely required for TREX occupancy at transcribed genes. Importantly, the Prp19 complex is necessary for full transcriptional activity. Taken together, we identify the Prp19 splicing complex as a novel transcription elongation factor that is essential for TREX occupancy at transcribed genes and that thus provides a novel link between transcription and messenger ribonucleoprotein (mRNP) formation.