Less than two percent of the human genome produces proteins, yet most of it is transcribed into RNA. Recent studies have been discovering a plethora of regulatory roles for noncoding RNA in diverse aspects of biology.

Dawning with growing interest in RNA is the insight that the stability, structure, and function of both coding and noncoding RNA revolves around how the transcribed nucleic acid chains are processed and outfitted with accessory chemical motifs that determine their unique destiny and action.

New research led by scientists at Columbia University, New York, is the first to reveal a specific modification of noncoding RNA pivotal in diversifying our seemingly infinite repertoire of antibodies against the relentless onslaught of infections and their accompanying antigenic insults.

Uttiya Basu, PhD, professor at the department of microbiology and immunology at Columbia University is the senior author of the study.

“RNA is rapidly becoming a very attractive vector for the delivery of vaccines and other therapeutics. Understanding how RNA therapeutic efficiency can be maximized is a major field of investigation. Our study provides evidence that RNA modifications are important factors to consider while developing RNA vaccines and therapeutics and underscores the vital and novel relationship of RNA modification and immunity,” says Uttiya Basu, PhD, professor at the department of microbiology and immunology at Columbia University and senior author on the study.

The study reported in this week’s Molecular Cell article “Mechanism of noncoding RNA associated N6-Methyladenosine recognition by an RNA processing complex during IgH DNA recombination” reveals modifying noncoding RNA with N6-methyladenosine (m6A)—a common modification in cellular DNA and protein-coding mRNA—allows the RNA processing complex called ‘RNA exosome’ to recognize a specific noncoding RNA (SµGLT) associated with the genomic site that encodes the heavy chain of antibodies (immunoglobulin H, IgH). This promotes a DNA shuffling mechanism central to the generation of diverse antibodies, called ‘class switch recombination’ (CSR), in antibody-producing B lymphocytes.

N6-methyladenosine modification of long noncoding RNAs expressed from the immunoglobulin heavy chain locus regulates DNA rearrangement events necessary for antibody gene diversity [Nair et al., 2021/ Molecular Cell]

“This study from Uttiya Basu’s laboratory takes us closer to understanding how we generate specific antibodies against the various antigens that we encounter. Here, the authors establish a crucial link between the N6-methyladenosine modification of long noncoding RNAs expressed from the immunoglobulin heavy chain locus and DNA rearrangement events that are necessary for antibody gene diversity.  As far as I am aware, this is the first study linking long noncoding RNA modifications with antibody gene diversity mechanisms,” says Sankar Ghosh, PhD, chairman and Silverstein and Hutt Family Professor at Columbia University’s Department of Microbiology & Immunology that includes Basu’s laboratory. Ghosh was not involved in the current study.

Two mechanisms are central in generating the nearly limitless diversity of antibodies in vertebrates: V(D)J recombination and class switch recombination (CSR). VDJ recombination is the process that selects and rearranges gene segments (called variable, joining, and diversity) that form the antigen-binding pocket of the antibody molecule, while CSR rearranges DNA that determines the carboxy-terminal constant region of the antibody molecule needed for optimal antibody function.

“Noncoding RNA surveillance pathway is important for promoting DNA recombination in B cells that leads to antibody gene diversity. It also prevents aberrant chromosomal alterations (translocations/mutations) that are the hallmark for lymphoma,” says Basu.

The RNA exosome is a part of the cellular noncoding RNA surveillance machinery that rapidly dismantles noncoding RNAs as they age. RNAs have specific lifespans at the end of which they must decay to prevent abnormal functions that may lead to disease, including cancers.  The noncoding RNA surveillance machinery determines the lifespan of noncoding RNAs that are associated with chromatin or floating in the nucleoplasm or cytosol.

Patricia J. Gearhart, PhD, deputy chief at the Laboratory of Molecular Biology and Immunology, National Institute on Aging (National Institutes of Health), whose work involves characterizing B cells in association with aging and atherosclerosis, and who is not involved in the current study, says, “It will be interesting to see if lymphoma patients have any mutations in the m6A pathway, which might link a preference for translocations over switch recombination. This could potentially serve as a diagnostic tool to predict cancer.”

“Noncoding RNA transcription that occurs at the antibody heavy and light chain loci are important for accessibility of DNA recombinase/mutator enzymes (RAG1/2 or AID) for V(D)J recombination, class switch recombination and somatic hypermutation. The accessibility is accomplished via mechanisms that are still under investigation but may be related to chromosomal organization, epigenetics, and DNA secondary structures,” says Basu.

Gearhart says, “Switch regions preceding genes for the immunoglobulin heavy chain are amazing G-rich DNA sequences that form long RNA-DNA hybrids during transcription. The structures then funnel in AID to create mutations and DNA strand breaks for recombination. Unanswered questions are: How is RNA removed from the template strand to allow AID to act on both DNA strands? And why are switch regions promiscuous partners for chromosomal translocations?”

The current comprehensive study establishes the convergence of m6A modification of noncoding RNA and RNA-exosome mediated processing of RNA in the catalysis of antibody diversity and the protection of genomic integrity.

“Here we show that methylation of noncoding RNA expressed from the immunoglobulin locus of B cells promotes RNA processing that is important for DNA recombination and isotype class switch recombination,” says Basu.

The authors show the RNA-exosome recognizes the SµGLT noncoding RNA through the interaction of an adaptor protein (MPP6) with a protein that reads the m6A modification (YTHDC1).  MPP6 and YTHDC1 make it possible for class switch recombination to occur by recruiting AID and the RNA exosome to SµGLT while it is being transcribed. The authors further show, blocking either m6A modification of SµGLT noncoding RNA or YTHDC1 decreases class switch recombination.

Lekha Nair, graduate student in Basu’s lab, is the lead author in this study.

Gearhart says, “Nair et al. propose switch region RNA processing is regulated by the RNA modification protein N6-methyladenosine. This methylation event marks the RNA to be recognized by reader proteins that recruit the RNA exosome to degrade the associated RNA and allow AID to act efficiently on both single-stranded top and bottom DNA strands. Thus, RNA processing, and not just transcription, is the major revelation of the paper.”

The enzyme that catalyzes m6A RNA modification is METTL3.  It is an essential gene for B cell development in the bone marrow and germinal centers of lymph nodes and its overexpression is seen in several cancers. In the current study, the researchers show METTL3 prevents abnormal DNA breaks at the immunoglobulin heavy chain site, preserving genomic stability. They also show, lack of METTL3 decreases CSR junctions and increases off-target DNA translocations, and the use of alternative DNA repair pathways.

“The right level of m6A modification on various RNAs (both messenger and noncoding RNAs) is necessary for preventing aberrant gene expression that leads to pathogenesis, including cancer. Here we show that when CSR does not occur properly, DNA breaks generated in the immunoglobulin heavy chain locus can have chromosomal translocations that could contribute towards B cell-associated cancers,” says Basu.

Overall, the evidence presented in the study suggests closely coordinated roles for the adaptor protein MPP6, the m6A reading protein YTHDC1 and METTL3-catalyzed m6A modification in regulating the processing of noncoding RNA that in turn controls DNA shuffling and the generation of antibody diversity in B cells.

Gearhart says, “In the absence of the protein, switch region RNA accumulates due to decreased exosome function, and AID is inefficiently recruited and produces aberrant beaks. Genome instability increases because the rogue breaks promote translocations. This tour de force work brings us closer to understanding the enigmatic role of switch regions in antibody diversity and DNA breaks.”

In future experiments, Basu’s team intends to explore other RNA modifications that play a role in generating antibody diversity.

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