The metazoan Integrator complex (INT) has important functions in the 3'-end processing of noncoding RNAs, including the uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA), and in the transcription of coding genes by RNA polymerase II. The INT contains at least 14 subunits, but its molecular mechanism of action is poorly understood, because currently there is little structural information about its subunits. The endonuclease activity of INT is mediated by its subunit 11 (IntS11), which belongs to the metallo-β-lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3'-end processing. IntS11 forms a stable complex with Integrator complex subunit 9 (IntS9) through their C-terminal domains (CTDs). Here, we report the crystal structure of the IntS9-IntS11 CTD complex at 2.1-Å resolution and detailed, structure-based biochemical and functional studies. The complex is composed of a continuous nine-stranded β-sheet with four strands from IntS9 and five from IntS11. Highly conserved residues are located in the extensive interface between the two CTDs. Yeast two-hybrid assays and coimmunoprecipitation experiments confirm the structural observations on the complex. Functional studies demonstrate that the IntS9-IntS11 interaction is crucial for the role of INT in snRNA 3'-end processing.

We recently demonstrated mammalian cells harbor NAD-capped mRNAs that are hydrolyzed by the DXO deNADding enzyme. Here we report the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targeting different RNAs. Crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 Å resolution provides exquisite insights into the molecular basis of the deNADding activity within the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to nutrient or environmental stress manifests changes in NAD-capped RNA levels that are selectively responsive to Nudt12 or DXO respectively, indicating an association of deNADding to cellular metabolism.

Integrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3′-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a β-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3′-end processing, maintenance of Cajal body structural integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator ‘cleavage module’ responsible for its endonucleolytic activity.

To investigate whether brassinosteroids (BRs) could be used to alleviate chill-induced inhibition of photosynthesis in cucumber (Cucumis sativus L) during chilling and subsequent recovery, the effects of exogenously applied 24-epibrassinolide (EBR) on gas exchange, chlorophyll fluorescence parameters, and antioxidant enzyme activity were studied. Cucumber plants were exposed to chilling under low light (12/8 o C and 100 μmol m -2 s -1 PPFD) for 3 days and then recovered under normal temperature and high irradiance (28/18 o C and 600 μmol m -2 s -1 PPFD) for 6 days. Chilling significantly decreased the net photosynthetic rate (P N ) and stomatal conductance (g s ), and increased rate of O 2˙formation and H 2 O 2 and malondialdehyde (MDA) content in cucumber leaves, but did not influence the optimal quantum yield of PSII (F v /F m ). Chilling also decreased the effective quantum yield of PSII photochemistry (Φ PSII ) and photochemical quenching (q P ), but induced an increase in nonphotochemical quenching (NPQ), and the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). High irradiance (600 μmol m -2 s -1 ) further aggravated the decrease in P N , g s , Φ PSII and q P , and enhanced the increase in reactive oxygen species (ROS) generation and accumulation in the first day of recovery after chilling. However, high irradiance induced a sharp decrease in F v /F m and NPQ, as well as the activities of SOD and APX on the first day of recovery. EBR pretreatment significantly alleviated chill-induced inhibition of photosynthesis during chilling stress and subsequent recovery period, which was mainly due to significant increases in g s , Φ PSII , q P and NPQ. EBR pretreatment also reduced ROS generation and accumulation, and increased the activities of SOD and APX during chilling and subsequent recovery. Those results suggest that EBR pretreatment alleviates the chill reduction in photosynthesis and accelerated the recovery rate mainly by increasing of the stomatal conductance, the efficiency of utilization and dissipation of leaf absorbed light, and the activity of the ROS scavenging system during chilling and subsequent recovery period. -light-adapted maximum fluorescence; F o -minimal fluorescence of dark-adapted state; F m -maximal fluorescence of dark-adapted state; F v /F m -optimal quantum yield of PSII; FM -fresh mass; g s -stomatal conductance; LT -low temperature; LTBR -low temperature; EBR-pretreatment; MDA -malondialdehyde; NT -normal temperature; NTBR -normal temperature/EBR-pretreatment; NPQ -nonphotochemical quenching; Φ PSII , -effective quantum yield of PSII photochemistry; P N -net photosynthetic rate; PPFD -photosynthetic photon flux density; q P -photochemical quenching coefficient; Rubisco -ribulose-1,5-bisphosphate carboxylase/oxygenase; ROS -reactive oxygen species; SOD -superoxide dismutase.

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