snRNA 3' End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis.

Uridine-rich small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play essential roles in splicing. The biogenesis of the majority of snRNAs involves 3' end endonucleolytic cleavage of the nascent transcript from the elongating DNA-dependent RNA ploymerase II. However, the protein factors responsible for this process remain elusive in plants. Here, we show that DEFECTIVE in snRNA PROCESSING 1 (DSP1) is an essential protein for snRNA 3' end maturation in Arabidopsis. A hypomorphic dsp1-1 mutation causes pleiotropic developmental defects, impairs the 3' end processing of snRNAs, increases the levels of snRNA primary transcripts (pre-snRNAs), and alters the occupancy of Pol II at snRNA loci. In addition, DSP1 binds snRNA loci and interacts with Pol-II in a DNA/RNA-dependent manner. We further show that DSP1 forms a conserved complex, which contains at least four additional proteins, to catalyze snRNA 3' end maturation in Arabidopsis. The catalytic component of this complex is likely the cleavage and polyadenylation specificity factor 73 kDa-I (CSPF73-I), which is the nuclease cleaving the pre-mRNA 3' end. However, the DSP1 complex does not affect pre-mRNA 3' end cleavage, suggesting that plants may use different CPSF73-I-containing complexes to process snRNAs and pre-mRNAs. This study identifies a complex responsible for the snRNA 3' end maturation in plants and uncovers a previously unknown function of CPSF73 in snRNA maturation.

Virtual screening of potential inhibitors from TCM for the CPSF30 binding site on the NS1A protein of influenza A virus.

Inhibition of CPSF30 function by the effector domain of influenza A virus of non-structural protein 1 (NS1A) protein plays a critical role in the suppression of host key antiviral response. The CPSF30-binding site of NS1A appears to be a very attractive target for the development of new drugs against influenza A virus. In this study, structure-based molecular docking was utilized to screen more than 30,000 compounds from a Traditional Chinese Medicine (TCM) database. Four drug-like compounds were selected as potential inhibitors for the CPSF30-binding site of NS1A. Docking conformation analysis results showed that these potential inhibitors could bind to the CPSF30-binding site with strong hydrophobic interactions and weak hydrogen bonds. Molecular dynamics simulations and MM-PBSA calculations suggested that two of the inhibitors, compounds 32056 and 31674, could stably bind to the CPSF30-binding site with high binding free energy. These two compounds could be modified to achieve higher binding affinity, so that they may be used as potential leads in the development of new anti-influenza drugs.

[Development of a yeast two-hybrid screen for selection of A/H1N1 influenza NS1 non-structural protein and human CPSF30 protein interaction inhibitors].

Influenza A/H1N1 virus-encoded nonstructural, or NS1, protein inhibits the 3'-end processing of cellular pre-mRNAs by binding the cellular protein: the 30-kDa subunit of CPSF (cleavage and polyadenylation specificity factor, CPSF30). CPSF30 binding site of the NS1 protein is a potential target for the development of drugs against influenza A/H1N1 virus. A yeast two-hybrid screening system was constructed and used for screening Chinese medicines that inhibit the interaction of the A/H1N1 flu NS1 protein and human CPSF30 protein. The NS1 gene of A/H1N1 virus was amplified by consecutive polymerase chain reaction (PCR), and the human CPSF30 gene of HeLa cell cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). Then the two gene fragments confirmed by sequencing were subcloned into the yeast expression vectors pGBKT7 and pGADT7, respectively. The two constructs, bait vector pGBKNS1 and prey vector pGADCPSF, were co-transformed into yeast AH109. The eight individual yeast colonies were picked and subjected to verification by PCR/gel electrophoresis. The inhibition of the NS1-CPSF30 interaction was allowed the identification of selective inhibitors. The four of more than thirty identified Chinese medicines, including 'Shuanghuanglian oral liquid', showed the strong inhibition of the NS1-CPSF30 interaction.

X-ray structures of NS1 effector domain mutants.

The influenza A virus nonstructural protein NS1 is a multifunctional dimeric protein that acts as a potent inhibitor of the host cellular antiviral state. The C-terminal effector domain of NS1 binds host proteins, including CPSF30, and is a target for the development of new antiviral drugs. Here we present crystallographic structures of two mutant effector domains, W187Y and W187A, of influenza A/Udorn/72 virus. Unlike wild-type, the mutants behave exclusively as monomers in solution based on gel filtration data and light scattering. The W187Y mutant is able to bind CPSF30 with a binding affinity close to the wild-type protein; that is, it retains a receptor site for aromatic ligands nearly identical to the wild-type. Therefore, this monomeric mutant protein could serve as a drug target for a high throughput inhibitor screening assays, since its binding pocket is unoccupied in solution and potentially more accessible to small molecule ligands.

The 73 kD subunit of the cleavage and polyadenylation specificity factor (CPSF) complex affects reproductive development in Arabidopsis.

The cleavage and polyadenylation specificity factor (CPSF) is an important multi-subunit component of the mRNA 3'-end processing apparatus in eukaryotes. The Arabidopsis genome contains five genes encoding CPSF homologues (AtCPSF160, AtCPSF100, AtCPSF73-I, AtCPSF73-II and AtCPSF30). These CPSF homologues interact with each other in a way that is analogous to the mammalian CPSF complex or their yeast counterparts, and also interact with the Arabidopsis poly(A) polymerase (PAP). There are two CPSF73 like proteins (AtCPSF73-I and AtCPSF73-II) that share homology with the 73 kD subunit of the mammalian CPSF complex. AtCPSF73-I appears to correspond to the functionally characterized mammalian CPSF73 and its yeast counterpart. AtCPSF73-II was identified as a novel protein with uncharacterized protein homologues in other multicellular organisms, but not in yeast. Both of the AtCPSF73 proteins are targeted in the nucleus and were found to interact with AtCPSF100. They are also essential since knockout or knockdown mutants are lethal. In addition, the expression level of AtCPSF73-I is critical for Arabidopsis development because overexpression of AtCPSF73-I is lethal. Interestingly, transgenic plants carrying an additional copy of the AtCPSF73-I gene, that is, the full-length cDNA under the control of its native promoter, appeared normal but were male sterile due to delayed anther dehiscence. In contrast, we previously demonstrated that a mutation in the AtCPSF73-II gene was detrimental to the genetic transmission of female gametes. Thus, two 73 kD subunits of the AtCPSF complex appear to have special functions during flower development. The important roles of mRNA 3'-end processing machinery in modulating plant development are discussed.