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In eukaryotes, ribosome assembly is a rate-limiting step in ribosomal biogenesis that takes place in a distinctive subnuclear organelle, the nucleolus. How ribosomes get assembled at the nucleolar site by forming initial preribosomal complexes remains poorly characterized. In this study, using several human and murine cell lines, we developed a method for isolation of native mammalian preribosomal complexes by lysing cell nuclei through mild sonication. A sucrose gradient fractionation of the nuclear lysate resolved several ribonucleoprotein (RNP) complexes containing rRNAs and ribosomal proteins. Characterization of the RNP complexes with MS-based protein identification and Northern blotting-based rRNA detection approaches identified two types of preribosomes we named here as intermediate preribosomes (IPRibs) and composed preribosome (CPRib). IPRib complexes comprised large preribosomes (105S to 125S in size) containing the rRNA modification factors and premature rRNAs. We further observed that a distinctive CPRib complex consists of an 85S preribosome assembled with mature rRNAs and a ribosomal biogenesis factor, Ly1 antibody-reactive (LYAR), that does not associate with premature rRNAs and rRNA modification factors. rRNA-labeling experiments uncovered that IPRib assembly precedes CPRib complex formation. We also found that formation of the preribosomal complexes is nutrient-dependent because the abundances of IPRib and CPRib decreased substantially when cells were either deprived of amino acids or exposed to an mTOR kinase inhibitor. These findings indicate that preribosomes form via dynamic and nutrient-dependent processing events and progress from an intermediate to a composed state during ribosome maturation.

Background Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown. Methods Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided. Results hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P Conclusion Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.

Background Reduced height-1 dwarfing alleles affect DELLA proteins belonging to a family of putative transcriptional regulators that modulate plant growth and development. The Arabidopsis thaliana genome encodes five DELLA proteins, whereas monocot plants, such as rice, barley, and wheat, each have a single DELLA protein. In wheat, wild-type Rht-B1a and Rht-D1a genes encode DELLA proteins and have many alleles that contain lesions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key part in the creation of high-yielding wheat varieties. Despite their fundamental roles in plant biology, until now, DELLA proteins in wheat have been mainly researched regarding the phenotypic effect of defective Rht mutants on yield-related traits, without studies on the underlying mechanisms. The RHT-1 protein has yet to be detected in wheat tissues, owing to a lack of appropriate molecular tools for characterization of RHT function and protein interactions in signal transduction. This study is focused on the production of a polyclonal antibody to the wheat RHT-D1A protein. Results To generate the anti-RHT-D1A antibody, we expressed and purified soluble 6xHis-tagged RHT-D1A. The purified recombinant RHT-D1A was injected into New Zealand white rabbits to generate polyclonal antiserum. The polyclonal anti-RHT-D1A antibody was purified by ammonium sulfate precipitation, followed by affinity chromatography on protein A–agarose beads. The purified polyclonal antibody was demonstrated to be effective in immunoblotting, western blot hybridization, and immunoprecipitation. In wheat seedling extracts, the polyclonal antibody recognized a protein with a molecular mass close to the predicted molecular weight of the endogenous RHT-D1A protein. We also demonstrated that RHT-D1A disappears in response to exogenous and endogenous gibberellic acid. Conclusion The purified polyclonal antibody raised against the recombinant RHT-D1A protein is sufficiently specific and sensitive and could be a useful tool for future insights into upstream and downstream components of DELLA-regulatory mechanisms in wheat plants.

In the Republic of Kazakhstan there are three types of areas with different cholera infection risk levels for people. Within cholera epidemiological surveillance the cholera strains are annually isolated from people and the environment. In this research, we studied the cholera strains isolated in Kazakhstan for 2013-2015 and they were selected for this study. All cholera strains were locally isolatedfrom people and the environment except one toxigenic imported strain of V. cholerae 01 lnaba which had genes of ctxAB, tcpA. The study showed that there were V cholerae strains which were atypical by their agglutination abilities and sensitivity to cholera phages. It can complicate the cholera laboratory diagnostics especially the diagnostics of V. cholerae О1 and for timely recognition of cholera it is necessary to carry out the diagnostics combined with molecular and genetic methods. For the study of antibiotic sensitivity or resistance we use eight antibacterial preparations. Cholera strains isolated in Kazakhstan did not have antibiotic resistance to the studied preparations. But the cholera strain brought from Pakistan was resistant to ciprofloxacin. The lack of strong resistance of local strains to the antibacterial drugs studied is encouraging in terms of conducting aetiotropic therapy. However, the possibility of entering of antibiotic-resistant strains into Kazakhstan obliges regularly to monitor strains for their sensitivity to antibacterial drugs.

Acute myeloid leukemia (AML) is a conglomerate of hematologic malignancies characterized by recurrent genetic and/or chromosomal aberrations. Recent development of targeted agents has bolstered our armamentarium of therapeutic options and improved outcomes for many patients. Acquiring deeper mechanistic understanding of myeloid leukemogenesis will provide a basis for development of even more therapeutic strategies and further improve patient outcomes. Our clinical data have revealed that overexpression of HNRNPK is a recurrent abnormality that occurs in upwards of 20% of AML cases at both the RNA and protein levels. Using bone marrow samples from a similar proportion of patients, we have recently discovered a supernumerary marker chromosome containing an extra copy of the HNRNPK locus that is not detectable with routine cytogenetic testing. We have further associated high hnRNP K protein levels with decreased overall survival in de novo AML, emphasizing the need to understand the role of hnRNP K in myeloid malignancy. To directly evaluate the oncogenic capacity of hnRNP K, we have overexpressed hnRNP K in murine fetal liver cells (FLCs). Using CyTOF and colony formation assays, we demonstrated that hnRNP K-overexpressing FLCs have altered differentiation potential and self-renewal capacity compared to empty vector controls in vitro. These findings are recapitulated in vivo, as murine recipients of hnRNP K-overexpressing FLCs develop myeloid lineage disease, often manifesting as fatal megakaryocytic leukemia. To elucidate a mechanism by which hnRNP K causes myeloid disease, we performed hnRNP K immunoprecipitation followed by mass spectrometry in an AML cell line and identified that hnRNP K preferentially interacts with translational machinery, ribosomal subunits, and proteins involved in RNA processing. In conjunction with data from our hnRNP K overexpression models that indicate overexpression of hnRNP K occurs primarily in the cytoplasm, we then performed hnRNP K-RNA immunoprecipitation followed by sequencing (RIP-Seq). We determined that hnRNP K interacts with the transcript of RUNX1-a master regulator of hematopoiesis and a critical player in a myriad of leukemias-including megakaryocytic leukemias like those observed in our mouse models. Using biochemical assays, we have demonstrated that hnRNP K directly binds to consensus sequences in the RUNX1 transcript, and ultimately alters RUNX1 translation. Indeed, mice exhibiting hnRNP K overexpression have increased protein levels of Runx1 in hematopoietic tissues. Our data demonstrate that hnRNP K overexpression drives myeloid malignancy. Currently, we are screening compounds that will disrupt the interaction between hnRNP K and RUNX1 in our efforts to further understand myeloid biology and ultimately improve outcomes for patients with these diseases. Disclosures No relevant conflicts of interest to declare.

hnRNP K is an RNA binding protein that controls a multitude of cellular processes and is aberrantly expressed in cancers. We have previously shown that hnRNP K functions as a haploinsufficient tumor suppressor in AML patients with 9q deletions. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon. We have recently discovered that hnRNP K overexpression in patients with diffuse large B-cell lymphoma correlates with dismal outcomes and directly resulted in the development of lymphomas in transgenic mice. To understand the mechanistic basis for the oncogenicity of hnRNP K overexpression and to identify therapeutic vulnerabilities, we performed RNA-sequencing, RNA immunoprecipitation following by sequencing (RIP-Seq), mass spectrometry, and polysome assays. We observed that hnRNP K regulates both global transcription and translational processes within the cell via modulation of 7SK and translation initiation proteins (such as the eIFs and PABP), respectively. Consequently, we hypothesized that aberrant hnRNP K expression primarily perturbs oncogenes with short half-lives. Mechanistically, we identified that hnRNP K binds to the RNA and regulates the expression of a plethora of critical oncogenes and tumor suppressors involved in hematologic malignancies such as c-Myc, RUNX1, and Cyclin D1. As proof of concept for clinical applications, we have demonstrated that hnRNP K-driven c-Myc overexpression renders tumors susceptible to bromodomain inhibition. Given that hnRNP K directs global transcription and translation, it is likely that hnRNP K overexpressing tumors will also be sensitive to transcriptional and translational inhibitors such as CDK9 inhibitors and omacetaxine mepesuccinate, respectively. However, since hnRNP K also regulates a plethora of additional cellular processes that extend far beyond mRNA and protein synthesis, there is a need to develop hnRNP K-specific inhibitors that will only target these activities. Thus, we have recently begun to identify small molecule compounds that can directly inhibit hnRNP K-RNA binding function on specific targets using an in vitro fluorescent binding assay. Using this assay, we are currently screening a library of 70,000 small molecule compounds to identify agents that can prevent hnRNP K-RNA interactions. In summary, we have established that hnRNP K is a bona fide oncogene that drives lymphomagenesis. Global analyses have revealed therapeutic vulnerabilities of hnRNP K overexpressing tumors. Furthermore, using our in vitro RNA binding assays, we anticipate identification of novel hnRNP K-specific inhibitors. Disclosures No relevant conflicts of interest to declare.