The common substrate structure for the functionally diverse Nudix protein superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving groups. The substrate specificity is known for less than 1% of the 29,400 known members. Most activities result in the release of an inorganic phosphate ion or of a product bearing a terminal phosphate moiety. Reactions have typically been monitored by a modification of the discontinuous Fiske–SubbaRow assay, which is relatively insensitive and slow. We report here the development of a continuous fluorescence assay that enables the rapid and accurate determination of substrate specificities in a 96-well format. We used this novel assay to confirm the reported substrate characterizations of MutT and NudD of Escherichia coli and to characterize DR_1025 of Deinococcus radiodurans and MM_0920 of Methanosarcina mazei. Novel findings enabled by the new assay include the following. First, in addition to the well-characterized hydrolysis of 8-oxo-dGTP at the α–β position, MutT cleaves at the β–γ phosphate bond at a rate of 3% of that recorded for hydrolysis at the α–β position. Second, MutT also catalyzes the hydrolysis of 5-methyl-dCTP. Third, 8-oxo-dGTP was observed to be the best substrate for DR_1025 of the 41 compounds screened.

Background: Nonsense mediated mRNA decay (NMD) is an RNA surveillance pathway that degrades aberrant transcripts harboring premature termination codons. This pathway, in conjunction with alternative splicing, regulates gene expression post-transcriptionally. Nearly all serine and arginine-rich (SR) proteins and many heterogeneous nuclear ribonucleoproteins (hnRNPs) produce isoforms that can be degraded by the NMD pathway. Many splicing factors have been reported to be regulated via alternative splicing coupled to NMD. However, it is still uncharacterized that to what extent NMD contributes to the regulation of splicing factors.Results: Here, we characterized a regulatory network of splicing factors through alternative splicing coupled to NMD. Based upon an extensive literature search, we first assembled a network that encompasses the current knowledge of splice factors repressing or activating the expression of other splicing factors through alternative splicing coupled to NMD. This regulatory network is limited, including just a handful of well-studied splicing factors. To gain a more global and less biased overview, we examined the splicing factor-mRNA interactions from public crosslinking-immunoprecipitation (CLIP)-seq data, which provides information about protein-RNA interactions. A network view of these interactions reveals extensive binding among splicing regulators. We also found that splicing factors bind more frequently to transcripts of other splicing factors than to other genes. In addition, many splicing factors are targets of NMD, and might be regulated via alternative splicing coupled to NMD, which is demonstrated by the significant overlap between the experimental network and eCLIP-network. We found that hierarchy of the splicing-factor interaction network differs from the hierarchy observed for transcription factors. Conclusion:The extensive interaction between splicing factors and transcripts of other splicing factors suggests that the potential regulation via alternative splicing coupled with NMD is widespread. The splicing factor regulation is fundamentally different from that of transcription factors.

To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini).We completed extensive in vitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild-type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity.AvrM14 is an extremely selective enzyme capable of removing the protective 5 0 cap from mRNA transcripts in vitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage in vitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild-type AvrM14, but not the catalytically dead version, suppressed immune-related reactive oxygen species production, altered the abundance of some circadian-rhythm-associated mRNA transcripts and reduced the hypersensitive cell-death response triggered by the flax disease resistance protein M1.To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with in vitro mRNA-decapping activity capable of interfering with plant immunity.

Activity assays performed with thin layer chromatography (TLC) TLC conditions were similar to the procedure described by Marini and Ipata (Marini & Ipata, 2007). Polyethyleneimine impregnated cellulose TLC plates (Flexible TLC Plates, Cellulose PEI F-254 from Selecto Scientific, 20 x 20 cm) were rinsed with 10 % NaCl (w/v), then rinsed twice with deionized water (Milli-Q, Millipore), and finally air dried. Next, the plates were cut into four 10 x10 cm squares. A typical reaction mixture contained 2.3 mM of a nucleoside triphosphate, 20 mM MgCl 2 , 50 mM CAPS, pH 10.2, and 0.01 mg/mL of TM0159. Each reaction mixture was incubated in a water bath at 40°C, 50°C, and 60°C. The reaction samples (2 µL) were spotted at the following time intervals: 5, 15, 30, 60, and 90 minutes. For reference, the TLC plates were spotted with 2 µL of standard solutions, such as 0.1 mM ITP and 0.1 mM IMP. All spots were eluted with 0.9 M LiCl, dried, and visualized in UV light at 254 nm with the help of a UV lamp (UV-LAMP-Spectroline model ENF-240C). To check whether TM0159 is also active at room temperature, an additional reaction was set up (2.3 mM ITP, 20 mM MgCl 2 , 50 mM CAPS, pH 10.2, and 0.01 mg/mL of TM0159) and kept at room temperature for 24 hours. After 24 hours, an aliquot was spotted on the TLC plate and visualized. TM0159 readily catalyzes the conversion of ITP to IMP at elevated temperatures. No conversion was detected at room temperature after 24 hours (Fig. S1). We also did not detect any conversion with the nucleoside triphosphate ATP. Figure S1: Photographs of two TLC plates that were used to monitor ITP hydrolysis catalyzed by TM0159 at different temperatures. Standard solutions (0.1 mM ITP and 0.1 mM IMP) were spotted on the plates as well: Lanes 6 & 13 ITP standard; lanes 7 & 14 IMP standard. Lanes 1-5: reaction temperature 40°C spotted after 5 min (lane 1), 15 min (lane 2), 30 min (lane 3), 60 min (lane 4), and 90 min (lane 5). Lanes 8-12: reaction temperature 50°C spotted after 5, 15, 30, 60, and 90 min. Lanes 15-19: reaction temperature 60°C spotted after 5, 15, 30, 60, and 90 min. Lane 20 contains a reaction sample incubated at room temperature for 24 hours.