Ig mRNAs Research Articles

Використання біоіндикаційного методу для оцінки забруднення атмосферного повітря важкими металами

The results of studies of chemical pollution (Pb, Zn) of perennial grasses—Elymus repens L., Artemisia absinthium L., and Hypericum perforatum L.—on background plots and within the areas of impact of industrial enterprises in Kropyvnytskyi (Ukraine) are presented. It is noted that chemical pollution is the most dangerous. The activity of enterprises is considered, in the result of which heavy metals enter the air. Testing of plants (stems and leaves) was carried out at a distance of 10–100 m from the enterprises. The content of heavy metals in the aboveground part of plants was analysed by atomic absorption spectrophotometer in the laboratory of the Department of Exploratory and Ecological Geochemistry, IGMR NAS of Ukraine. The comparison of sorption properties of different plants in the same phases of development allowed plants with bioindication capabilities to be identified. The results of the analysis showed that plants most effectively accumulate the maximum amount of heavy metals from the atmospheric air at the end of the summer dormancy phase (second half of August). At this time the plant requires the minimum amount of nutrients that it takes from the soil. Stems and leaves of perennial grasses actively accumulate aerosols and fine dust on their surface due to plant secretions with the formation of a mucous film that actively adsorbs heavy metal ions. The background, minimum, and maximum content of heavy metals in plants is determined. Comparing the content of heavy metals in plant samples in the background and study plots, it was found that E. repens near industrial enterprises is the most effective bioindicator of air pollution. In order to determine the level of atmospheric air pollution using bioindication methods, the index of plant contamination (Sр) was proposed, which is the ratio of concentration coefficient of heavy metals and their amount. An assessment scale for atmospheric air pollution with heavy metals has been developed based on the contamination index (Sр) of bioindicator plants: the degree of pollution is 1–3—weak, 3–6—medium, 6–9—strong, more than 9—very strong. The proposed coefficients allow for conducting a situational assessment of atmospheric air pollution with heavy metals using bioindication methods.

FAM46C controls antibody production by the polyadenylation of immunoglobulin mRNAs and inhibits cell migration in multiple myeloma.

FAM46C, frequently mutated in multiple myeloma (MM), has recently been shown to encode a non‐canonical poly(A) polymerase (ncPAP). However, its target mRNAs and its role in MM pathogenesis remain mostly unknown. Using CRISPR‐Cas9 technology and gene expression analysis, we found that the inactivation of FAM46C in MM down‐regulates immunoglobulins (Igs) and several mRNAs encoding ER‐resident proteins, including some involved in unfolded protein response and others that affect glycosylation. Interestingly, we show that FAM46C expression is induced during plasma cell (PC) differentiation and that Ig mRNAs encoding heavy and light chains are substrates of the ncPAP, as revealed by poly(A) tail‐length determination assays. The absence of the ncPAP results in Ig mRNA poly(A) tail‐shortening, leading to a reduction in mRNA and protein abundance. On the other hand, loss of FAM46C up‐regulates metastasis‐associated lncRNA MALAT1 and results in a sharp increase in the migration ability. This phenotype depends mainly on the activation of PI3K/Rac1 signalling, which might have significant therapeutic implications. In conclusion, our results identify Ig mRNAs as targets of FAM46C, reveal an important function of this protein during PC maturation to increase antibody production and suggest that its role as a tumour suppressor might be related to the inhibition of myeloma cell migration.

FAM46C Controls Antibody Production By the Polyadenylation of Ig mRNAs and Inhibits Cell Migration in Multiple Myeloma

FAM46C, which is frequently mutated in multiple myeloma (MM), has recently been shown to encode a non-canonical poly(A) polymerase (ncPAP). However, its target mRNAs and its role in MM pathogenesis remain largely unknown. Using CRISPR-Cas9 technology and gene expression analysis we found that inactivation of FAM46C in MM downregulates immunoglobulins (Igs) and several mRNAs encoding ER-resident proteins, including some involved in unfolded protein response (UPR), such as PDIA6, ERP44 and EDEM2, and others that affect glycosylation, such as SSR4, AGA and DDOST. We also found that FAM46C knockout cells exhibited lower ER stress than controls after treatment with tunicamycin, as indicated by RT-PCR analysis of spliced/unspliced forms of XBP1 mRNA. Interestingly, we show that FAM46C expression is induced during PC differentiation and that Ig mRNAs encoding secretory heavy and light chains are direct substrates of the ncPAP, as revealed by poly(A) tail-length determination assays. The absence of the ncPAP results in Ig mRNA poly(A) tail-shortening, leading to a reduction in mRNA and protein abundance. On the other hand, loss of FAM46C upregulated metastasis-associated lncRNA MALAT1 and results in a sharp increase in the migration ability of MM cells. This phenotype depends mainly on the activation of PI3K/Rac1 signaling in FAM46C knockout cells since treatment with specific inhibitors substantially reduces cell mobility. This finding may have significant therapeutic implications.In conclusion, our results identify, for the first time, Ig mRNAs as targets of FAM46C, reveal an important function of this protein during PC maturation to increase antibody production, and suggest that its role as a tumor suppressor might be related with the inhibition of cell migration in MM. DisclosuresGarcia-Sanz:Affimed: Research Funding.

Expression of immunoglobulin G in human podocytes, and its role in cell viability and adhesion.

Podocyte injury occurs during the initiation and development of numerous forms of glomerular disease, and antibodies targeting podocytes have become a biomarker for diagnosis and monitoring treatment response. Accumulating evidence has suggested that immunoglobulin (Ig) is expressed in non-B lineage cells, including epithelial cancer cells, myeloid cells and several types of normal cells. The main aim of the present study was to ascertain the expression of IgG in human podocytes and to determine its potential role in cellular bioactivity. The present study detected positive staining for IgG heavy chain (Igγ) and its subtype γ4, and the light chains κ and λ in the cytoplasm or on the membrane by immunofluorescence. In addition, positive bands were detected for Igγ, γ1, γ3, γ4, κ and λ in the lysates of a podocyte cell line by western blotting. Mass spectrometry confirmed IgG1 as an intact tetramer in the culture supernatant. Constant region transcripts of Igγ, γ1, γ3, γ4, κ and λ were identified by reverse transcription-polymerase chain reaction, and DNA sequencing of these transcripts revealed 96–99% similarity with Ig mRNAs in the National Center for Biotechnology Information database. Compared with the diverse gene rearrangements from B cell-derived Ig, podocyte-derived Ig exhibited conservative V(D)J patterns in the variable regions of Igγ and κ chains. Furthermore, the present study investigated the mechanism underlying IgG production in these cells by examining the expression of recombination activating gene (RAG)1, RAG2 and activation-induced cytidine deaminase. The expression levels of these proteins suggested that podocyte-derived Ig and traditional Ig may be generated in a similar manner. Furthermore, small interfering RNA-mediated downregulation of IgG expression reduced podocyte viability and adhesive capabilities. These findings suggested that IgG is expressed in podocytes and that this expression may be associated with podocyte function. Due to its potential biological and clinical significance, this phenomenon warrants further investigation.

Effects of AhR ligands on the production of immunoglobulins in purified mouse B cells

The aryl hydrocarbon receptor (AhR) has been shown to play important roles in the immune system, and contributions of AhR ligands to the differentiation and functions of Th17/Treg cells have recently been established. However, it has not been fully clarified whether AhR plays roles in B cell differentiation and functions. The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly potent AhR agonist, was reported to suppress the production of immunoglobulin M (IgM) in a transformed mouse B cell line. However, TCDD exhibits high toxicity toward cells and has unknown activities except for its action as an AhR agonist. In the present study, we tried to clarify how an endogenously generated AhR agonist affects mouse B cell differentiation and functions in terms of the direct effects on the expression of Ig subclasses in purified mouse B cells stimulated with an anti-CD40 antibody and interleukin-4. The AhR agonist 2-(1'H-indole-3'- carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), which is derived via tryptophan metabolism, suppressed the expression of not only IgM, but also IgG1 and IgE. ITE was also found to suppress the expression of secreted-type Ig mRNAs and plasma cell-specific genes. These findings indicate that the endogenous AhR agonist suppresses B cell differentiation into Ig-secreting plasma cells.

Sequence Determination of the Heavy-Chain Constant Region in Four Immunoglobulin Classes of Mongolian Gerbils (<i>Meriones unguiculatus</i>)

We determined partial cDNA sequences of four immunoglobulin (Ig) classes-IgM, IgG1, IgE, and IgA-of Mongolian gerbil (Meriones unguiculatus). Each deduced Ig heavy-chain constant (IGHC) region-Cµ, Cγ1, Cε, and Cα-is structurally similar to its counterparts in the mouse and rat, and phylogenetic analysis suggests that the gerbil Igs are evolutionarily close to their counterparts. In spite of the high sequence homology to the other rodent Cγ sequences, the gerbil Cγ1 sequence differs from our previously reported Cγ2. This result indicates that the gerbil has at least two IgG subclasses. These four gerbil IGHC cDNA sequences will be useful for determining gerbil Ig isotypes and examining the expression of gerbil Ig mRNAs in response to parasitic and bacterial infections.

Effect of hypoosmotic pressure on cell growth and antibody production in recombinant Chinese hamster ovary cell culture.

To determine the response of recombinant Chinese hamster ovary (rCHO) cells subjected to hypoosmotic pressure, rCHO cells (CS13*-1.0) producing a chimeric antibody were cultivated in the hypoosmolar medium resulting from NaCl subtraction. At hypoosmotic pressure, CS13*-1.0 cells displayed decreased specific growth rate (mu) and increased specific antibody productivity (q (Ab)).When the medium osmolality was decreased from 300 mOsm kg(-1)(physiological osmolality) to 150 mOsm kg(-1), mu was decreased by 68% and q (Ab) was increased by 128%. To understand the mechanism of enhanced q (Ab) resulting from hypoosmotic pressure, cellular responses of cells in the exponential phase of growth were observed at the transcription level. Total cytoplasmic RNA content per cell at 150 mOsm kg(-1) was increased by 140%, compared with that at 300 mOsm kg(-1). On a per mug RNA basis, immunoglobulin (Ig) mRNA levels at 150 mOsm kg(-1) were comparable to those at 300 mOsm kg(-1), indicating that hypoosmotic pressure did not lead to the preferential transcription of Ig mRNAs. Taken together, the data obtained here suggest that the increase in total RNA pool is primarily responsible for the enhanced q (Ab) of CS13*-1.0 cells subjected to hypoosmotic pressure.

Hyperosmotic pressure enhances immunoglobulin transcription rates and secretion rates of KR12H-2 transfectoma.

When subjected to hyperosmotic pressure resulting from NaCl addition, KR12H-2 transfectoma, like most hybridomas, displayed a decrease in specific growth rate (mu) and an increase in specific antibody productivity (q(Ab)). Elevation of medium osmolality from 285 to 425 mOsm/kg decreased mu by 20%, while it increased q(Ab) by 376%. Although cell mass also increased at higher osmolality, it was not the main factor in increasing q(Ab). Hyperosmotic pressure was found to enhance transcription levels of immunoglobulin (Ig) mRNAs preferentially, compared with non-IgG mRNA. The transcription levels of both heavy chain (HC) and light chain (LC) mRNAs were enhanced as much as q(Ab). This result suggests that enhanced q(Ab) at higher osmolality was mainly due to enhanced transcription levels of Ig mRNA. However, these increased transcription levels of Ig mRNAs were not due to the enhanced stability of Ig mRNA. In fact, the stability of Ig mRNAs decreased at higher osmolality. Elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg decreased the half-lives of HC and LC mRNAs by 37% and 36%, respectively. A simple mathematical model revealed that transcription rates of Ig mRNAs increased by more than 476% at 425 mOsm/kg. These elevated transcription levels could, in turn, increase the translation rates of Ig polypeptides. However, the translation rates of Ig polypeptides were not enhanced as much as the transcription levels of Ig mRNAs and q(Ab). The elevation of osmolality from 285 mOsm/kg to 425 mOsm/kg increased HC and LC mRNA specific translation rates by 172% and 240%, respectively. Taken together, the data suggest that (1) enhanced q(Ab) of KR12H-2 transfectoma at higher osmolality is due to elevated transcription rates of Ig mRNAs and expedited post-translational processing of Ig, and (2) antibody secretion by KR12H-2 transfectoma is most likely controlled at the level of Ig translation, particularly HC translation.

Developmental regulation of immunoglobulin mRNA processing and the IgA response: establishing a paradigm.

IgA, which is protective at mucosal sites, is derived from memory B cells that develop in the organized lymphoid tissue of the gastrointestinal tract and subsequently mature to plasma cells in the lamina propria. Similarly to B cells expressing other isotypes, the maturation of IgA-expressing B cells is associated with both an increase in the steady-state level of immunoglobulin mRNA and the ratio of secreted to membrane forms of mRNA, which differ in 3' terminus. In contrast to B cells expressing other isotypes, at all stages in the development of an IgA response, the secreted form of alpha mRNA predominates. In this article, studies on the general features of IgA B cell development, mechanisms regulating 3' terminus usage of Ig mRNAs, and isotype-specific regulation of 3' terminus usage particularly in regard to alpha mRNA are discussed.

Ku80 is required for immunoglobulin isotype switching.

Isotype switching is the DNA recombination mechanism by which antibody genes diversify immunoglobulin effector functions. In contrast to V(D)J recombination, which is mediated by RAG1, RAG2 and DNA double-stranded break (DSB) repair proteins, little is known about the mechanism of switching. We have investigated the role of DNA DSB repair in switch recombination in mice that are unable to repair DSBs due to a deficiency in Ku80 (Ku80(-/-)). B-cell development is arrested at the pro-B cell stage in Ku80(-/-) mice because of abnormalities in V(D)J recombination, and there are no mature B cells. To reconstitute the B-cell compartment in Ku80(-/-) mice, pre-rearranged VB1-8 DJH2 (mu i) and V3-83JK2 (kappa i) genes were introduced into the Ku80(-/-) background (Ku80(-/-)mu i/+kappa i/+). Ku80(-/-)mu i/+ kappai/+ mice develop mature mIgM+ B cells that respond normally to lipopolysaccharide (LPS) or LPS plus interleukin-4 (IL-4) by producing specific germline Ig constant region transcripts and by forming switch region-specific DSBs. However, Ku80(-/-)mu i/+kappa i/+ B cells are unable to produce immunoglobulins of secondary isotypes, and fail to complete switch recombination. Thus, Ku80 is essential for switch recombination in vivo, suggesting a significant overlap between the molecular machinery that mediates DNA DSB repair, V(D)J recombination and isotype switching.

Alternative poly(A) site selection in complex transcription units: means to an end?

Many genes have been described and characterized which result in alternative polyadenylation site use at the 3'-end of their mRNAs based on the cellular environment. In this survey and summary article 95 genes are discussed in which alternative polyadenylation is a consequence of tandem arrays of poly(A) signals within a single 3'-untranslated region. An additional 31 genes are described in which polyadenylation at a promoter-proximal site competes with a splicing reaction to influence expression of multiple mRNAs. Some have a composite internal/terminal exon which can be differentially processed. Others contain alternative 3'-terminal exons, the first of which can be skipped in some cells. In some cases the mRNAs formed from these three classes of genes are differentially processed from the primary transcript during the cell cycle or in a tissue-specific or developmentally specific pattern. Immunoglobulin heavy chain genes have composite exons; regulated production of two different Ig mRNAs has been shown to involve B cell stage-specific changes in trans -acting factors involved in formation of the active polyadenylation complex. Changes in the activity of some of these same factors occur during viral infection and take-over of the cellular machinery, suggesting the potential applicability of at least some aspects of the Ig model. The differential expression of a number of genes that undergo alternative poly(A) site choice or polyadenylation/splicing competition could be regulated at the level of amounts and activities of either generic or tissue-specific polyadenylation factors and/or splicing factors.

Regulation of transcription of the germline immunoglobulin alpha constant region gene.

Different classes of antibodies serve different effector functions and therefore the production of different classes must be regulated during an immune response. Numerous recent studies support the accessibility model for the regulation of heavy chain class switch recombination, as heavy chain (CH) genes to which cells will switch have been shown to be hypomethylated and to be trarrscriptionally active (reviewed in Esser and Radbruch, 1990). Each of the unrearranged CH genes has been shown to be transcribed prior to switch recombination to that particular CH gene. The structures of all the CH gene germline transcripts are identical, i.e. they are initiated 5’ to the switch (S) regions and are transcribed in the same direction as and terminate near the same poly(A) sites as mature Ig mRNAs (Fig. 1). An exon 5’ to the S regions (I or germline exon) is spliced to the normal acceptor site for mature mRNAs. The germline RNAs tend to have multiple initiation sites probably due to the fact that they do not have TATA boxes and at least one (germline α RNA) has multiple splice donor sites for the I exon.

Alternative promoters and exons, somatic mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma.

The most common translocation in human lymphoma, the t(14;18)(q32;q21), generates heterogeneous 4.2-7.2 kb Bcl-2-immunoglobulin (Ig) chimeric mRNAs resulting from alternative Bcl-2 5' exons and varied Ig 3' untranslated regions (UT). The normal human Bcl-2 gene has a three exon structure with an untranslated first exon, a facultative 220 bp intron I, but an enormous 370 kb intron II. S1 protection and primer extension analysis defined initiation sites in exon II associated with classic promoter elements and a decanucleotide (ATG-CAAAGCA) homologous with Ig variable region enhancers. Multiple initiation sites were also found in a GC-rich region with Sp1 binding motifs in exon I. Most t(14;18) breakpoints cluster within the 3' UT of Bcl-2 implicating that event in gene deregulation. The Bcl-2 gene introduced into the Ig constant (C gamma) locus of SU-DHL-6 displayed somatic mutation. While Bcl-2--Ig mRNAs demonstrated an unaltered 2.5 h half-life, the Bcl-2--Ig gene revealed an inappropriately high rate of transcription for a mature B-cell. This indicates the translocated Bcl-2 allele has escaped normal control mechanisms.

Membrane-bound ribosomes of myeloma cells. V. Subcellular distribution of immunoglobulin mRNA molecules.

The subcellular distribution of the most abundant mRNA sequences, particularly those of the immunoglobulin heavy (Ig H) and light (IG L) chain mRNA sequences, of MOPC 21 (P3K) mouse myeloma cells has been examined by translating the mRNA of various subcellular fractions in a messenger-dependent reticulocyte lysate (MDL) and by identifying Ig products with the use of a specific antiserum. Analyses of the distribution of the mRNA template activity and the translation products by SDS polyacrylamide gel electrophoresis reveal that approximately 85% of the mRNA present in the free ribosomal fraction is incorporated into polysomes and that the remainder is present as mRNP particles. On the endoplasmic reticulum (ER) the mRNA is found entirely in polysomes. In general, the size class of free (F) and membrane-bound (MB) polysomes corresponds to the size of their translation products. Thus, mRNAs coding Ig H (5.0 x 10(5) daltons in size) and Ig L (2.5 x 10(5) daltons in size) are incorporated into polysomes formed of 12 and 6 ribosomes, respectively. About 10% of the Ig mRNAs are not bound to membranes. A third of these are associated with mRNPs and the remainder incorporated into F polysomes of the same size as the Ig-synthesizing MB polysomes.

The synthesis and processing of the messenger RNAs specifying heavy and light chain immunoglobulins in MPC-11 cells

The nuclear precursors of the immunoglobulin messenger RNAs of MPC-11 cells were characterized with respect to size, amount per cell and extent of polyadenylation. These cells produce three Ig mRNAs: a 1.8 kb component coding for a γ 2b heavy chain (H mRNA), a 1.2 kb mRNA coding for a κ light chain (L mRNA) and a 0.8 kb mRNA coding for the constant region portion of the κ light chain (L 1 mRNA). To identify the pre-mRNAs without ambiguity, we constructed recombinant DNA plasmids containing H and L cDNA sequences, and used the cloned cDNAs as hybridization probes for analysis of steady state nuclear RNA and in DNA excess hybridization experiments with pulse-labeled nuclear RNA. The nuclear molecules containing Ig sequences consist of an 11 kb component (H 1), which we believe to be the primary transcript of the H gene, 5.3 kb (L 1), and 3.3 kb (L 2) components, which seem to be primary transcripts of the L and L 1 genes, components corresponding to mature size H, L and L 1 mRNAs, and several intermediatesized components which include the processing derivatives. The precursor role of these nuclear molecules was established by studies of their labeling kinetics and by appropriate pulse-chase experiments. All the pre-mRNA species including H 1, L 1 and L 2 contain poly(A), thus suggesting that polyadenylation is an early event in the processing of these mRNAs. The MPC-11 cell contains about 30,000 and 40,000 cytoplasmic H and L mRNA molecules, respectively, which must be produced within one cell generation (∼24 hr). In comparison, the nucleus contains about 100–150 molecules of total pre-mRNA and only about 10–15 molecules of presumptive primary transcripts for each of these Ig species. These values indicate very rapid transcription rates (>20 transcripts per min) and exceptionally fast processing rates (∼0.5 min for the primary transcripts and ∼5 min for overall nuclear processing) for the Ig mRNAs. Thus rapid transcription and processing, together with high cytoplasmic stability, account for the high abundance of Ig mRNAs in the myeloma cell.

Translation of human immunoglobulin heavy chain mRNA in vitro

MOUSE immunoglobulin (Ig) mRNAs have been the subject of intensive study. Purification of Ig light chain mRNAs from myeloma or plasmacytoma cells has been monitored by translation in vitro, and studies have been directed towards sequence determination and gene titration Moreover, partial purification of Ig heavy chain mRNA has also been achieved1–5. No comparable work on the Ig mRNA of any other species has yet been published. We report here the preparation, fractionation and translation in vitro of mRNA from cells of a cloned normal human B lymphocytic cell line. RPMI 1788 (ref. 6). grown in tissue culture. Human Ig μ chains were identified in the translation product by specific immunoprecipitation and electrophoretic mobility in sodium dodecyl sulphate (SDS)-urea polyacrylamide gels.