NoLogo: a new statistical model highlights the diversity and suggests new classes of Crm1-dependent nuclear export signals.

BACKGROUND: Crm1-dependent Nuclear Export Signals (NESs) are clusters of alternating hydrophobic and non-hydrophobic amino acid residues between 10 to 15 amino acids in length. NESs were largely thought to follow simple consensus patterns, based on which they were categorized into 6-10 classes. However, newly discovered NESs often deviate from the established consensus patterns. Thus, identifying NESs within protein sequences remains a bioinformatics challenge. RESULTS: We describe a probabilistic representation of NESs using a new generative model we call NoLogo that can account for a large diversity of NESs. Using this model to predict NESs, we demonstrate improved performance over PSSM and GLAM2 models, but do not achieve the performance of the state-of-the-art NES predictor LocNES. Our findings illustrate that over 30% of NESs are best described by novel NES classes rather than the 6-10 classes proposed by current/existing models. Finally, many NESs have additional hydrophobic residues either upstream or downstream of the canonical four residues, suggesting possible functionality. CONCLUSION: Applying the NoLogo model highlights the observation that NESs are more diverse than previously appreciated. Our work questions the practice of assigning each NES to one of several predefined NES classes. Finally, our analysis suggests a novel and testable biophysical perspective on interaction between Crm1 receptor and Crm1-dependent NESs.

The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) develops through distinct precursor lesions, including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). However, genetic features resulting in IPMN-associated PDA (IPMN-PDA) versus PanIN-associated PDA (PanIN-PDA) are largely unknown. Here we find that loss of Brg1, a core subunit of SWI/SNF chromatin remodelling complexes, cooperates with oncogenic Kras to form cystic neoplastic lesions that resemble human IPMN and progress to PDA. Although Brg1-null IPMN-PDA develops rapidly, it possesses a distinct transcriptional profile compared with PanIN-PDA driven by mutant Kras and hemizygous p53 deletion. IPMN-PDA also is less lethal, mirroring prognostic trends in PDA patients. In addition, Brg1 deletion inhibits Kras-dependent PanIN development from adult acinar cells, but promotes Kras-driven preneoplastic transformation in adult duct cells. Therefore, this study implicates Brg1 as a determinant of context-dependent Kras-driven pancreatic tumorigenesis and suggests that chromatin remodelling may underlie the development of distinct PDA subsets.

Directed differentiation of human pluripotent stem cells along the pancreatic endocrine lineage.

Many research groups are engaged in using human pluripotent stem cells (hPSCs) to generate surrogate pancreatic ß-cells for transplantation into diabetic patients. However, to our knowledge, there is no report on the successful generation of glucose-responsive insulin-producing ß-cells from hPSCs in vitro. Below, we outline a method that is based on published protocols as well as our own experience by which one can differentiate hPSCs along the pancreatic lineage to generate insulin-producing ß-cell-like cells. The protocol, which spans five distinct stages, is an attempt to recapitulate the derivation of pancreatic ß-cells in vitro as they form in the developing embryo. We included details on materials and techniques, suggest ways to customize it to your hPSC line of choice, added notes on how to monitor and analyze the cells during differentiation, and indicate what results can be expected.

Regulatory evolution in proteins by turnover and lineage-specific changes of cyclin-dependent kinase consensus sites.

Evolutionary change in gene regulation is a key mechanism underlying the genetic component of organismal diversity. Here, we study evolution of regulation at the posttranslational level by examining the evolution of cyclin-dependent kinase (CDK) consensus phosphorylation sites in the protein subunits of the pre-replicative complex (RC). The pre-RC, an assembly of proteins formed during an early stage of DNA replication, is believed to be regulated by CDKs throughout the animals and fungi. Interestingly, although orthologous pre-RC components often contain clusters of CDK consensus sites, the positions and numbers of sites do not seem conserved. By analyzing protein sequences from both distantly and closely related species, we confirm that consensus sites can turn over rapidly even when the local cluster of sites is preserved, consistent with the notion that precise positioning of phosphorylation events is not required for regulation. We also identify evolutionary changes in the clusters of sites and further examine one replication protein, Mcm3, where a cluster of consensus sites near a nucleocytoplasmic transport signal is confined to a specific lineage. We show that the presence or absence of the cluster of sites in different species is associated with differential regulation of the transport signal. These findings suggest that the CDK regulation of MCM nuclear localization was acquired in the lineage leading to Saccharomyces cerevisiae after the divergence with Candida albicans. Our results begin to explore the dynamics of regulatory evolution at the posttranslational level and show interesting similarities to recent observations of regulatory evolution at the level of transcription.

CDK phosphorylation of a novel NLS-NES module distributed between two subunits of the Mcm2-7 complex prevents chromosomal rereplication.

Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (pre-RCs) at replication origins to prevent inappropriate rereplication. In Saccharomyces cerevisiae, one of these mechanisms promotes the net nuclear export of a pre-RC component, the Mcm2-7 complex, during S, G2, and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raises the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.