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.

Small Nuclear Ribonucleoprotein Polypeptide A-Mediated Alternative Polyadenylation of STAT5B during Th1 Cell Differentiation.

T cells are activated and differentiated into Th cells depending on the rapid and accurate changes in the cell transcriptome. In addition to changes in mRNA expression, the sequences of many transcripts are altered by alternative splicing and alternative polyadenylation (APA). We profiled the APA sites of human CD4+ T cell subsets with high-throughput sequencing and found that Th1 cells harbored more genes with shorter tandem 3' untranslated regions (UTRs) than did naive T cells. We observed that STAT5B, a key regulator of Th1 differentiation, possessed three major APA sites and preferred shorter 3' UTRs in Th1 cells. In addition, small nuclear ribonucleoprotein polypeptide A (SNRPA) was found to bind directly to STAT5B 3' UTR and facilitate its APA switching. We also found that p65 activation triggered by TCR signaling could promote SNRPA transcription and 3' UTR shortening of STAT5B. Thus we propose that the APA switching of STAT5B induced by TCR activation is mediated by SNRPA.

The role of alternative polyadenylation in the antiviral innate immune response.

Alternative polyadenylation (APA) is an important regulatory mechanism of gene functions in many biological processes. However, the extent of 3' UTR variation and the function of APA during the innate antiviral immune response are unclear. Here, we show genome-wide poly(A) sites switch and average 3' UTR length shortens gradually in response to vesicular stomatitis virus (VSV) infection in macrophages. Genes with APA and mRNA abundance change are enriched in immune-related categories such as the Toll-like receptor, RIG-I-like receptor, JAK-STAT and apoptosis-related signalling pathways. The expression of 3' processing factors is down-regulated upon VSV infection. When the core 3' processing factors are knocked down, viral replication is affected. Thus, our study reports the annotation of genes with APA in antiviral immunity and highlights the roles of 3' processing factors on 3' UTR variation upon viral infection.

Dynamic Regulation of Tandem 3' Untranslated Regions in Zebrafish Spleen Cells during Immune Response.

Alternative polyadenylation (APA) has been found to be involved in tumorigenesis, development, and cell differentiation, as well as in the activation of several subsets of immune cells in vitro. Whether APA takes place in immune responses in vivo is largely unknown. We profiled the variation in tandem 3' untranslated regions (UTRs) in pathogen-challenged zebrafish and identified hundreds of APA genes with ∼ 10% being immune response genes. The detected immune response APA genes were enriched in TLR signaling, apoptosis, and JAK-STAT signaling pathways. A greater number of microRNA target sites and AU-rich elements were found in the extended 3' UTRs than in the common 3' UTRs of these APA genes. Further analysis suggested that microRNA and AU-rich element-mediated posttranscriptional regulation plays an important role in modulating the expression of APA genes. These results indicate that APA is extensively involved in immune responses in vivo, and it may be a potential new paradigm for immune regulation.

Is transcription the dominant force during dynamic changes in gene expression?

Dynamic changes in gene expression punctuate lymphocyte development and are a characteristic of lymphocyte activation. A prevailing view has been that these changes are driven by DNA transcription factors, which are the dominant force in gene expression. Accumulating evidence is challenging this DNA centric view and has highlighted the prevalence and dynamic nature of RNA handling mechanisms. Alternative splicing and differential polyadenylation appear to be more widespread than first thought. Changes in mRNA decay rates also affect the abundance of transcripts and this mechanism may contribute significantly to gene expression. Additional RNA handling mechanisms that control the intracellular localization of mRNA and association with translating ribosomes are also important. Thus, gene expression is regulated through the coordination of transcriptional and post-transcriptional mechanisms. Developing a more "RNA centric" view of gene expression will allow a more systematic understanding of how gene expression and cell function are integrated.

Increased phosphorylation of the carboxyl-terminal domain of RNA polymerase II and loading of polyadenylation and cotranscriptional factors contribute to regulation of the ig heavy chain mRNA in plasma cells.

B cells produce Ig H chain (IgH) mRNA and protein, primarily of the membrane-bound specific form. Plasma cells produce 20- to 50-fold higher amounts of IgH mRNA, most processed to the secretory specific form; this shift is mediated by substantial changes in RNA processing but only a small increase in IgH transcription rate. We investigated RNA polymerase II (RNAP-II) loading and phosphorylation of its C-terminal domain (CTD) on the IgG2a H chain gene, comparing two mouse cell lines representing B (A20) and plasma cells (AxJ) that express the identical H chain gene whose RNA is processed in different ways. Using chromatin immunoprecipitation and real-time PCR, we detected increased RNAP-II and Ser-2 and Ser-5 phosphorylation of RNAP-II CTD close to the IgH promoter in plasma cells. We detected increased association of several 3' end-processing factors, ELL2 and PC4, at the 5' end of the IgH gene in AxJ as compared with A20 cells. Polymerase progress and factor associations were inhibited by 5,6-dichlorobenzimidazole riboside, a drug that interferes with the addition of the Ser-2 to the CTD of RNAP-II. Taken together, these data indicate a role for CTD phosphorylation and polyadenylation/ELL2/PC4 factor loading on the polymerase in the choice of the secretory poly(A) site for the IgH gene.