The Investigation of Cleavage Factor IM by Crystallographic and Biochemical Techniques

RNA maturation involves several steps prior to export of the mRNA out of the nucleus and translation in the cytoplasm. PremRNA 3’end processing is one of such steps, and comprises the endonucleolytic cleavage and polyadenylation of the 3’end of the premRNA. These two steps involve more than 14 processing factors that coordinate multiple proteinprotein and proteinRNA interactions necessary to coordinate efficient cleavage and polyadenylation. To date, many of these interactions have been investigated biochemically and require additional structural characterization both to confirm and highlight key residues involved in substrate contacts. Further structural characterization will also open investigation into the mechanism of 3’end processing by providing structural insight into the coordination of multiple binding components. The cleavage factor Im, CF Im, is a component of the 3’end processing machinery and plays an important role early, during endonucleolytic cleavage, and additionally to increase polyadenylation efficiency and regulate poly(A) site recognition. CF Im is composed of a small 25 kDa subunit, CF Im25, and a large, either 58 kDa, 68 kDa, or 72 kDa subunit. The 25 kDa subunit of CF Im interacts with both the RNA and other processing factors such as the poly(A) polymerase, Clp1, and the larger subunit of CF Im. It is our goal to crystallize CF Im25 alone and in complex with one of its interacting partners to better understand CF Im25 contributions to premRNA 3’end processing. The structural investigation of CF Im25 and its binding partners has accomplished four major objectives: 1) Characterized the crystal structure of CF Im25 alone and bound to diadenosine tetraphosphate, 2) Provided insight into the oligomeric state of the CF Im complex, 3) Determined the binding properties of the Nudix domain of CF Im25 and its function in 3’end processing, 4) Further characterize the interactions between CF Im25 and PAP, CF Im68, and Clp1. These results demonstrate CF Im25 is a dimer both in solution and in the crystal suggesting that it is likely to be a dimer in the CF Im complex. The nucleotide binging capability of CF Im25 has no apparent role in 3’end processing in vitro but may provide a function outside of 3’end processing or may directly be involved in RNA recognition. The additional investigation of complex interactions with the 25 kDa subunit of CF Im25 suggests that although these factors interact during the 3’end processing event additional mechanisms may play a role in stabilizing those interactions

Crystal structure of the 25 kDa subunit of human cleavage factor Im

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing

A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family

The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality

NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis

Genome-wide Analysis of Pre-mRNA 3′ End Processing Reveals a Decisive Role of Human Cleavage Factor I in the Regulation of 3′ UTR Length

Through alternative polyadenylation, human mRNAs acquire longer or shorter 30 untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To under- stand the dynamics of polyadenylation site usage, we performed transcriptome-wide mapping of both binding sites of 30 end processing factors CPSF- 160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64t, CF Im25, CF Im59, and CF Im68 and 30 end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF-64/CstF-64t and CFIm proteins have much higher positional specificity compared to CPSF components. Knockdown of CF Im68 induced a systematic use of proximal polyade- nylation sites, indicating that changes in relative abundance of a single 30 end processing factor can modulate the length of 30 untranslated regions across the transcriptome and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF Im68 knockdown