Human RPF1 and ESF1 in Pre-rRNA Processing and the Assembly of Pre-Ribosomal Particles: A Functional Study

Ribosome biogenesis is essential for the functioning of living cells. In higher eukaryotes, this multistep process is tightly controlled and involves a variety of specialized proteins and RNAs. This pool of so-called ribosome biogenesis factors includes diverse proteins with enzymatic and structural functions. Some of them have homologs in yeast S. cerevisiae, and their function can be inferred from the structural and biochemical data obtained for the yeast counterparts. The functions of human proteins RPF1 and ESF1 remain largely unclear, although RPF1 has been recently shown to participate in 60S biogenesis. Both proteins have drawn our attention since they contribute to the early stages of ribosome biogenesis, which are far less studied than the later stages. In this study, we employed the loss-of-function shRNA/siRNA-based approach to the human cell line HEK293 to determine the role of RPF1 and ESF1 in ribosome biogenesis. Downregulating RPF1 and ESF1 significantly changed the pattern of RNA products derived from 47S pre-rRNA. Our findings demonstrate that RPF1 and ESF1 are associated with different pre-ribosomal particles, pre-60S, and pre-40S particles, respectively. Our results allow for speculation about the particular steps of pre-rRNA processing, which highly rely on the RPF1 and ESF1 functions. We suggest that both factors are not directly involved in pre-rRNA cleavage but rather help pre-rRNA to acquire the conformation favoring its cleavage

Human nucleolar protein SURF6/RRP14 participates in early steps of pre-rRNA processing.

The biogenesis of ribosomes requires tightly controlled transcription and processing of pre-rRNA which comprises ribosomal RNAs forming the core of large and small ribosomal subunits. Early steps of the pre-rRNA processing and assembly of the ribosomal subunits require a large set of proteins that perform folding and nucleolytic cleavage of pre-rRNAs in the nucleoli. Structure and functions of proteins involved in the pre-rRNA processing have been extensively studied in the budding yeast S. cerevisiae. Functional characterization of their human homologues is complicated by the complexity of mammalian ribosomes and increased number of protein factors involved in the ribosomal biogenesis. Homologues of human nucleolar protein SURF6 from yeast and mouse, Rrp14 and Surf6, respectively, had been shown to be involved in the early steps of pre-rRNA processing. Rrp14 works as RNA chaperone in complex with proteins Ssf1 and Rrp15. Human SURF6 knockdown and overexpression were used to clarify a role of SURF6 in the early steps of pre-rRNA processing in human cell lines HeLa and HTC116. By analyzing the abundance of the rRNA precursors in cells with decreased level or overexpression of SURF6, we demonstrated that human SURF6 is involved in the maturation of rRNAs from both small and large ribosomal subunits. Changes in the SURF6 level caused by knockdown or overexpression of the protein do not result in the death of HeLa cells in contrast to murine embryonic fibroblasts, but significantly alter the distribution of cells among the phases of the cell cycle. SURF6 knockdown in both p53 sufficient and p53 deficient HCT116 human cancer cells results in elongation of G0/G1 and shortening of G2/M phase. This surprising result suggests p53 independence of SURF6 effects on the cell cycle and possible multiple functions of SURF6. Our data point to the shift from pathway 1 to pathway 2 of the rRNA biogenesis caused by the SURF6 knockdown and its likely association with p53 pathway

Human Natural Antibodies Recognizing Glycan Galβ1-3GlcNAc (LeC)

International audienceThe level of human natural antibodies of immunoglobulin M isotype against LeC in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors' blood using LeC-Sepharose (LeC is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with LeC, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10-9 M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galβ1-3GlcNAcβ disaccharide, indicates that the target epitope of anti-LeC antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan

Specificity of human natural antibodies referred to as anti-Tn

International audienceTo understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα−Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1−3Galβ1−3(4)GlcNAcβ. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα−OSer and do not bind a wide variety of GalNAcα−OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a con-formationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs

Human natural antibodies recognizing glycan galβ1-3glcnac (LeC)

The level of human natural antibodies of immunoglobulin M isotype against LeC in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors’ blood using LeC-Sepharose (LeC is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with LeC, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10−9 M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galβ1-3GlcNAcβ disaccharide, indicates that the target epitope of anti-LeC antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan. © 2020 by the authors. Licensee MDPI, Basel, Switzerland