Nuclear Proximity of Mtr4 to RNA Exosome Restricts DNA Mutational Asymmetry.

The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and senataxin) with the noncoding RNA processing function of RNA exosome determine the strand-specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development.

The RNA Exosome Syncs IAV-RNAPII Transcription to Promote Viral Ribogenesis and Infectivity.

The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery.

Mechanism of noncoding RNA-associated N6-methyladenosine recognition by an RNA processing complex during IgH DNA recombination.

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SµGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N6-methyladenosine (m6A) RNA modification drives recognition and 3' end processing of SµGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m6A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SµGLT. Direct suppression of m6A modification of SµGLT or of m6A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m6A modification, and m6A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells.

Fam72a enforces error-prone DNA repair during antibody diversification.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

Targeting IgE polyadenylation signal with antisense oligonucleotides decreases IgE secretion and plasma cell viability.

BACKGROUND: Allergy regroups numerous complex and various diseases classified as IgE-dependent or non-IgE-dependent hypersensitivities. IgEs are expressed as membrane and secreted forms by B cells and plasma cells, respectively. In IgE-mediated hypersensitivity, IgE secretion and binding to the high-affinity IgE receptor FcεRI on effector cells are responsible for the onset of allergic symptoms; in contrast, surface IgE expression as a B-cell receptor is barely detectable. OBJECTIVE: Our aim was to test an innovative antisense approach to reducing IgE secretion. METHODS: We designed an antisense oligonucleotide (ASO) targeting the polyadenylation signal of human secreted IgE to redirect IgE transcript polyadenylation from the secreted form to the membrane form. ASO treatments were performed on B cells from transgenic mice expressing humanized IgE (InEps mice), as well as on human primary B cells and myeloma cells. In vivo ASO delivery was tested by using an InEps mouse model. RESULTS: We demonstrated that treatment with a morpholino ASO targeting the secreted IgE polyadenylation signal drastically decreased IgE secretion and inversely increased membrane IgE mRNA expression. In addition, ASO treatment induced apoptosis of IgE-expressing U266 myeloma cells, and RNA sequencing revealed attenuation of their plasma cell phenotype. Remarkably, systemic administration of an ASO coupled with Pip6a as an arginine-rich cell-penetrating peptide decreased IgE secretion in vivo. CONCLUSION: Altogether, this ASO strategy could be an effective way to decrease IgE secretion and allergic symptoms in patients with IgE-dependent allergies, and it could also promote allergen tolerance through apoptosis of IgE+ antibody-secreting cells.

Roles of G4-DNA and G4-RNA in Class Switch Recombination and Additional Regulations in B-Lymphocytes.

Mature B cells notably diversify immunoglobulin (Ig) production through class switch recombination (CSR), allowing the junction of distant "switch" (S) regions. CSR is initiated by activation-induced deaminase (AID), which targets cytosines adequately exposed within single-stranded DNA of transcribed targeted S regions, with a specific affinity for WRCY motifs. In mammals, G-rich sequences are additionally present in S regions, forming canonical G-quadruplexes (G4s) DNA structures, which favor CSR. Small molecules interacting with G4-DNA (G4 ligands), proved able to regulate CSR in B lymphocytes, either positively (such as for nucleoside diphosphate kinase isoforms) or negatively (such as for RHPS4). G4-DNA is also implicated in the control of transcription, and due to their impact on both CSR and transcriptional regulation, G4-rich sequences likely play a role in the natural history of B cell malignancies. Since G4-DNA stands at multiple locations in the genome, notably within oncogene promoters, it remains to be clarified how it can more specifically promote legitimate CSR in physiology, rather than pathogenic translocation. The specific regulatory role of G4 structures in transcribed DNA and/or in corresponding transcripts and recombination hereby appears as a major issue for understanding immune responses and lymphomagenesis.

RNA processing mechanisms contribute to genome organization and stability in B cells.

RNA processing includes post-transcriptional mechanisms controlling RNA quality and quantity to ensure cellular homeostasis. Noncoding (nc) RNAs that are regulated by these dynamic processes may themselves fulfill effector and/or regulatory functions, and recent studies demonstrated the critical role of RNAs in organizing both chromatin and genome architectures. Furthermore, RNAs can threaten genome integrity when accumulating as DNA:RNA hybrids, but could also facilitate DNA repair depending on the molecular context. Therefore, by qualitatively and quantitatively fine-tuning RNAs, RNA processing contributes directly or indirectly to chromatin states, genome organization, and genome stability. B lymphocytes represent a unique model to study these interconnected mechanisms as they express ncRNAs transcribed from key specific sequences before undergoing physiological genetic remodeling processes, including V(D)J recombination, somatic hypermutation, and class switch recombination. RNA processing actors ensure the regulation and degradation of these ncRNAs for efficient DNA repair and immunoglobulin gene remodeling while failure leads to B cell development alterations, aberrant DNA repair, and pathological translocations. This review highlights how RNA processing mechanisms contribute to genome architecture and stability, with emphasis on their critical roles during B cell development, enabling physiological DNA remodeling while preventing lymphomagenesis.

A p53 defect sensitizes various stages of B cell development to lymphomagenesis in mice carrying an IgH 3' regulatory region-driven c-myc transgene.

Although c-myc is classically described as the driving oncogene in Burkitt's lymphoma (BL), deregulation and mutations of c-myc have been reported in multiple solid tumors and in other mature B cell malignancies such as mantle cell lymphoma (MCL), myeloma, and plasma cell lymphoma (PCL). After translocation into the IgH locus, c-myc is constitutively expressed under the control of active IgH enhancers. Those located in the IgH 3' regulatory region (3'RR) are master control elements of class switch recombination and of the transcriptional burst associated with plasma cell differentiation. c-myc-3'RR mice are prone to lymphomas with rather homogeneous, most often BL-like, phenotypes with incomplete penetrance (75% tumor incidence) and long latencies (10-12 mo). To reproduce c-myc-induced mature B cell lymphomagenesis in the context of an additional defect often observed in human lymphomas, we intercrossed c-myc-3'RR with p53(+/-) mice. Double transgenic c-myc-3'RR/p53(+/-) mice developed lymphoma with short latency (2-4 mo) and full penetrance (100% tumor incidence). The spectrum of B lymphomas occurring in c-myc-3'RR/p53(+/-) mice was widened, including nonactivated (CD43(-)) BL, activated (CD43(+)) BL, MCL-like lymphoma, and PCL, thus showing that 3'RR-mediated deregulation of c-myc can promote various types of B lymphoproliferation in cells that first acquired a p53 defect. c-myc/p53(+/-) mice closely reproduce many features of BL, MCL, and PCL and provide a novel and efficient model to dissect the molecular events leading to c-myc-induced lymphomagenesis and an important tool to test potential therapeutic agents on malignant B cells featuring various maturation stages.

Unique repetitive nucleic acid structures mirror switch regions in the human IgH locus.

Immunoglobulin (Ig) genes carry the unique ability to be reshaped in peripheral B lymphocytes after these cells encounter a specific antigen. B cells can then further improve their affinity, acquire new functions as memory cells and eventually end up as antibody-secreting cells. Ig class switching is an important change that occurs in this context, thanks to local DNA lesions initiated by the enzyme activation-induced deaminase (AID). Several cis-acting elements of the Ig heavy (IgH) chain locus make it accessible to the AID-mediated lesions that promote class switch recombination (CSR). DNA repeats, with a non-template strand rich in G-quadruplexes (G4)-DNA, are prominent cis-targets of AID and define the so-called "switch" (S) regions specifically targeted for CSR. By analyzing the structure of the human IgH locus, we uncover that abundant DNA repeats, some with a putative G4-rich template strand, are additionally present in downstream portions of the IgH coding genes. These like-S (LS) regions stand as 3' mirror-images of S regions and also show analogies to some previously reported repeats associated with the IgH locus 3' super-enhancer. A regulatory role of LS repeats is strongly suggested by their specific localization close to exons encoding the membrane form of Ig molecules, and by their conservation during mammalian evolution.

Attempts to evaluate locus suicide recombination and its potential role in B cell negative selection in the mouse.

Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain (IgH) locus under control of its 3' cis-regulatory region (3'RR). The 3'RR is itself transcribed and can undergo "locus suicide recombination" (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined. Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3'RR. Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies. Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro, this study suggests that LSR may contribute to the elimination of self-reactive B cells.

Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells.

BACKGROUND: While most antibody-based therapies use IgG because of their well-known biological properties, some functional limitations of these antibodies call for the development of derivatives with other therapeutic functions. Although less abundant than IgG in serum, IgA is the most abundantly produced Ig class in humans. Besides the specific targeting of its dimeric form to mucosal areas, IgA was shown to recruit polymorphonuclear neutrophils against certain targets more efficiently than does IgG1. DESIGN AND METHODS: In this study, we investigated the various pathways by which anti-tumor effects can be mediated by anti-CD20 IgA against lymphoma cells. RESULTS: We found that polymeric human IgA was significantly more effective than human IgG1 in mediating direct killing or growth inhibition of target cells in the absence of complement. We also demonstrated that this direct killing was able to indirectly induce the classical pathway of the complement cascade although to a lesser extent than direct recruitment of complement by IgG. Recruitment of the alternative complement pathway by specific IgA was also observed. In addition to activating complement for lysis of lymphoma cell lines or primary cells from patients with lymphoma, we showed that monomeric anti-CD20 IgA can effectively protect mice against tumor development in a passive immunization strategy and we demonstrated that this protective effect may be enhanced in mice expressing the human FcαRI receptor on their neutrophils. CONCLUSIONS: We show that anti-CD20 IgA antibodies have original therapeutic properties against lymphoma cells, with strong direct effects, ability to recruit neutrophils for cell cytotoxicity and even recruitment of complement, although largely through an indirect way.

Specific impairment of proximal tubular cell proliferation by a monoclonal κ light chain responsible for Fanconi syndrome.

BACKGROUND: Fanconi syndrome (FS) is a rare renal disorder featuring proximal tubule dysfunction that may occur following tubular reabsorption of a monoclonal light chain (LC), in patients with multiple myeloma. FS may precede the recognition of multiple myeloma by several years. In most cases, crystalline inclusions of monoclonal κ LCs are observed within the lysosomes of proximal tubular cells (PTCs) and probably participate in their functional alteration. METHODS: To investigate the mechanism implicated in proximal tubule dysfunction, we compared the effects of κ LC-CHEB obtained from a patient with myeloma-associated FS to those of control κ LC-BON obtained from a patient without evidence of FS, on the viability and proliferation of two different PTC lines. RESULTS: Our data suggest that the tubular atrophy in myeloma-associated FS does not result from increased apoptosis of PTCs, but from their impaired capacity to proliferate and renew. Indeed, in vitro incubation of cultured PTCs with physiological amounts of the nephrotoxic κ LC-CHEB was sufficient to cause a depression in DNA synthesis and in cell proliferation. This effect was observed neither with control κ LC-BON nor in the absence of κ LC. CONCLUSIONS: The reduced turnover of PTCs may affect tubular repair and regeneration. In addition, the reduced proliferation of myeloma cells producing the same monoclonal κ LC might explain the frequent association of FS with smoldering multiple myeloma.

Identification of RNA-DNA Hybrids Associated with R-Loops at the IgH Switch Sequence in Activated B Cells.

During transcription and replication, R-loops that contain RNA-DNA hybrids are generated across numerous genomic loci and contribute to many biological events. Using S9.6, a monoclonal antibody against RNA-DNA hybrids, accelerated the study of R-loop biology. An outpouring of recent studies has implicated various contributions of R-loop in physiological cellular functions. Earlier studies using nondenaturing sodium bisulfite probing also supported existence of DNA-RNA hybrids formation in mammalian cells. In activated B cells, RNA-DNA hybrids formation at IgH gene locus of B cells is crucial for class switch recombination that ensure the proper effector function of the antibody. Here, we describe the identification of R-loops associated with the IgH locus using RNA-DNA hybrid immunoprecipitation sequencing and nondenaturing sodium bisulfite probing. This will be helpful for future studies of R-loops status on whole genome as well as on IgH locus in B cells.

RNA exosome drives early B cell development via noncoding RNA processing mechanisms.

B cell development is linked to successful V(D)J recombination, allowing B cell receptor expression and ultimately antibody secretion for adaptive immunity. Germline noncoding RNAs (ncRNAs) are produced at immunoglobulin (Ig) loci during V(D)J recombination, but their function and posttranscriptional regulation are incompletely understood. Patients with trichohepatoenteric syndrome, characterized by RNA exosome pathway component mutations, exhibit lymphopenia, thus demonstrating the importance of ncRNA surveillance in B cell development in humans. To understand the role of RNA exosome in early B cell development in greater detail, we generated mouse models harboring a B cell-specific cre allele (Mb1cre), coupled to conditional inversion-deletion alleles of one RNA exosome core component (Exosc3) or RNase catalytic subunits (Exosc10 or Dis3). We noticed increased expression of RNA exosome subunits during V(D)J recombination, whereas a B cell developmental blockade at the pro-B cell stage was observed in the different knockout mice, overlapping with a lack of productive rearrangements of VDJ genes at the Ig heavy chain (Igh). This unsuccessful recombination prevented differentiation into pre-B cells, with accumulation of ncRNAs and up-regulation of the p53 pathway. Introduction of a prearranged Igh VDJ allele partly rescued the pre-B cell population in Dis3-deficient cells, although V-J recombination defects were observed at Ig light chain kappa (Igκ), preventing subsequent B cell development. These observations demonstrated that the RNA exosome complex is important for Igh and Igκ recombination and establish the relevance of RNA processing for optimal diversification at these loci during B cell development.

UnAIDed Class Switching in Activated B-Cells Reveals Intrinsic Features of a Self-Cleaving IgH Locus.

Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) gene diversification in germinal center B-cells. From its first description, it was considered as mandatory for class switch recombination (CSR), and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood with regards the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence, based on deep-sequencing approaches, showing that this dogma is not absolute in both human and mouse B lymphocytes. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo "on-target" cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus.

Noncoding RNA processing by DIS3 regulates chromosomal architecture and somatic hypermutation in B cells.

Noncoding RNAs are exquisitely titrated by the cellular RNA surveillance machinery for regulating diverse biological processes. The RNA exosome, the predominant 3' RNA exoribonuclease in mammalian cells, is composed of nine core and two catalytic subunits. Here, we developed a mouse model with a conditional allele to study the RNA exosome catalytic subunit DIS3. In DIS3-deficient B cells, integrity of the immunoglobulin heavy chain (Igh) locus in its topologically associating domain is affected, with accumulation of DNA-associated RNAs flanking CTCF-binding elements, decreased CTCF binding to CTCF-binding elements and disorganized cohesin localization. DIS3-deficient B cells also accumulate activation-induced cytidine deaminase-mediated asymmetric nicks, altering somatic hypermutation patterns and increasing microhomology-mediated end-joining DNA repair. Altered mutation patterns and Igh architectural defects in DIS3-deficient B cells lead to decreased class-switch recombination but increased chromosomal translocations. Our observations of DIS3-mediated architectural regulation at the Igh locus are reflected genome wide, thus providing evidence that noncoding RNA processing is an important mechanism for controlling genome organization.

ERK1/2 phosphorylation predicts survival following anti-PD-1 immunotherapy in recurrent glioblastoma.

Only a subset of recurrent glioblastoma (rGBM) responds to anti-PD-1 immunotherapy. Previously, we reported enrichment of BRAF/PTPN11 mutations in 30% of rGBM that responded to PD-1 blockade. Given that BRAF and PTPN11 promote MAPK/ERK signaling, we investigated whether activation of this pathway is associated with response to PD-1 inhibitors in rGBM, including patients that do not harbor BRAF/PTPN11 mutations. Here we show that immunohistochemistry for ERK1/2 phosphorylation (p-ERK), a marker of MAPK/ERK pathway activation, is predictive of overall survival following adjuvant PD-1 blockade in two independent rGBM patient cohorts. Single-cell RNA-sequencing and multiplex immunofluorescence analyses revealed that p-ERK was mainly localized in tumor cells and that high-p-ERK GBMs contained tumor-infiltrating myeloid cells and microglia with elevated expression of MHC class II and associated genes. These findings indicate that ERK1/2 activation in rGBM is predictive of response to PD-1 blockade and is associated with a distinct myeloid cell phenotype.

Effects of senataxin and RNA exosome on B-cell chromosomal integrity.

Loss of function of senataxin (SETX), a bona-fide RNA/DNA helicase, is associated with neuronal degeneration leading to Ataxia and Ocular Apraxia (AOA) in human patients. SETX is proposed to promote transcription termination, DNA replication, DNA repair, and to unwind deleterious RNA:DNA hybrids in the genome. In all the above-mentioned mechanisms, SETX unwinds transcription complex-associated nascent RNA which is then degraded by the RNA exosome complex. Here we have used B cells isolated from a SETX mutant mouse model and compared genomic instability and immunoglobulin heavy chain locus (IgH) class switch recombination (CSR) to evaluate aberrant and programmed genomic rearrangements, respectively. Similar to RNA exosome mutant primary B cells, SETX mutant primary B cells display genomic instability but a modest decrease in efficiency of CSR. Furthermore, knockdown of Setx mRNAs from CH12-F3 B-cell lines leads to a defect in IgA CSR and accumulation of aberrant patterns of mutations in IgH switch sequences. Given that SETX mutant mice do not recapitulate the AOA neurodegenerative phenotype, it is possible that some aspects of SETX biology are rescued by redundant helicases in mice. Overall, our study provides new insights into the role of the SETX/RNA exosome axis in suppressing genomic instability so that programmed DNA breaks are properly orchestrated.

Noncoding RNA transcription alters chromosomal topology to promote isotype-specific class switch recombination.

B cells undergo two types of genomic alterations to increase antibody diversity: introduction of point mutations into immunoglobulin heavy- and light-chain (IgH and IgL) variable regions by somatic hypermutation (SHM) and alteration of antibody effector functions by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). SHM and CSR require the B cell-specific activation-induced cytidine deaminase (AID) protein, the transcription of germline noncoding RNAs, and the activity of the 3' regulatory region (3'RR) super-enhancer. Although many transcription regulatory elements (e.g., promoters and enhancers) reside inside the IgH and IgL sequences, the question remains whether clusters of regulatory elements outside IgH control CSR. Using RNA exosome-deficient mouse B cells where long noncoding RNAs (lncRNAs) are easily detected, we identified a cluster of three RNA-expressing elements that includes lncCSRIgA (that expresses lncRNA-CSRIgA). B cells isolated from a mouse model lacking lncRNA-CSRIgA transcription fail to undergo normal levels of CSR to IgA both in B cells of the Peyer's patches and grown in ex vivo culture conditions. lncRNA-CSRIgA is expressed from an enhancer site (lncCSRIgA ) to facilitate the recruitment of regulatory proteins to a nearby CTCF site (CTCFlncCSR) that alters the chromosomal interactions inside the TADlncCSRIgA and long-range interactions with the 3'RR super-enhancer. Humans with IgA deficiency show polymorphisms in the lncCSRIgA locus compared with the normal population. Thus, we provide evidence for an evolutionarily conserved topologically associated domain (TADlncCSRIgA) that coordinates IgA CSR in Peyer's patch B cells through an lncRNA (lncRNA-CSRIgA) transcription-dependent mechanism.