ABSTRACT
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.
Subject(s)
Adenosine/analogs & derivatives , B-Lymphocytes/metabolism , Exosome Multienzyme Ribonuclease Complex/metabolism , Immunoglobulin Heavy Chains/metabolism , RNA 3' End Processing , RNA, Long Noncoding/metabolism , Recombination, Genetic , Adenosine/metabolism , Animals , B-Lymphocytes/immunology , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Exosome Multienzyme Ribonuclease Complex/genetics , Female , Genomic Instability , HEK293 Cells , Humans , Immunoglobulin Class Switching , Immunoglobulin Heavy Chains/genetics , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Mice, Knockout , RNA, Long Noncoding/genetics , RNA, Untranslated/genetics , RNA, Untranslated/metabolismABSTRACT
Purpose: Second harmonic generation signals (SHG) are emitted preferentially from collagenous tissue structures and have been used to evaluate photochemically-induced (CXL) crosslinking changes in the cornea. Since therapeutic tissue crosslinking (TXL) using sodium hydroxymethylglycinate (SMG) of the sclera is a potential treatment for high myopia, we explored the use of SHG microscopy to evaluate the effects. Methods: Single sub-Tenon's (sT) injections (400 µL) using SMG (40-400 mM) were made at the equatorial 12 o'clock position of the right eye of cadaveric rabbit heads (n = 16 pairs). After 3.5 hours, confocal microscopy (CM) was performed using 860 nm two-photon excitation and 400 to 450 nm emission. Pixel density and fiber bundle "waviness" analyses were performed on the images. Crosslinking effects were confirmed using thermal denaturation (Tm) temperature. Comparison experiments with riboflavin photochemical crosslinking were done. Results: Therapeutic tissue crosslinking localization studies indicated that crosslinking changes occurred at the site of injection and in adjacent sectors. Second harmonic generation signals revealed large fibrous collagenous bundled structures that displayed various degrees of waviness. Histogram analysis showed a nearly 6-fold signal increase in 400 mM SMG over 40 mM. This corresponded to a ΔTm = 13°C for 400 mM versus ΔTm = 4°C for 40 mM. Waviness analysis indicated increased fiber straightening as a result of SMG CXL. Conclusions: Second harmonic generation signal intensity and fiber bundle waviness is altered by scleral tissue crosslinking using SMG. These changes provide insights into the macromolecular changes that are induced by therapeutic crosslinking technology and may provide a method to evaluate connective tissue protein changes induced by scleral crosslinking therapies.
Subject(s)
Collagen/metabolism , Cross-Linking Reagents/administration & dosage , Myopia/drug therapy , Sarcosine/analogs & derivatives , Sclera/pathology , Animals , Disease Models, Animal , Injections , Microscopy, Confocal , Myopia/diagnosis , Myopia/metabolism , Rabbits , Sarcosine/administration & dosage , Sclera/drug effectsABSTRACT
Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.
