Discovering and Mapping the Modified Nucleotides That Comprise the Epitranscriptome of mRNA.

An important mechanism of gene expression regulation is the regulated modification of nucleotides in messenger RNA (mRNA). These modified nucleotides affect mRNA translation, stability, splicing, and other processes. A cluster of nucleotide modifications is found adjacent to the mRNA cap structure and another set can be found internally within transcripts. The most prominent modifications are methylations of adenosine to form either N 6-methyladenosine (m6A), an internal modified nucleotide, or N 6,2'-O-dimethyladenosine (m6Am), which is found exclusively at the first templated nucleotide of certain mRNAs. In addition, other rare modified nucleotides have been identified and together these form the epitranscriptomic code of mRNA. In the case of some modified nucleotides, the presence, location, or abundance is a subject of debate. Here, we review the methods that enable the discovery of modified nucleotides and how these approaches can be used to map epitranscriptomic modifications in mRNA.

Mapping m6A at Individual-Nucleotide Resolution Using Crosslinking and Immunoprecipitation (miCLIP).

N 6 -methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current m6A mapping approaches localize m6A residues to 100-200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here, we describe a method for using anti-m6A antibodies to induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Then, we describe how to use these mutational signatures to map m6A residues at nucleotide resolution. Taken together, our protocol allows for high-throughput detection of individual m6A residues throughout the transcriptome.

Reversible methylation of m6Am in the 5' cap controls mRNA stability.

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2'-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.

N(6)-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current mapping approaches localize m6A residues to transcript regions 100-200 nt long but cannot identify precise m6A positions on a transcriptome-wide level. Here we developed m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) and used it to demonstrate that antibodies to m6A can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA cross-linking and reverse transcription. We found that these antibodies similarly induced mutational signatures at N(6),2'-O-dimethyladenosine (m6Am), a modification found at the first nucleotide of certain mRNAs. Using these signatures, we mapped m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identified small nucleolar RNAs (snoRNAs) as a new class of m6A-containing non-coding RNAs (ncRNAs).

mRNA metabolism and neuronal disease.

To serve as templates for translation eukaryotic mRNAs undergo an elaborate processing and maturation pathway. In eukaryotes this process comprises the synthesis of mRNA precursors, their processing and transport to the site of translation and eventually their decay. During the entire life cycle, mRNAs interact with distinct sets of trans-acting factors that determine their fate at any given phase of gene expression. Recent studies have shown that mutations in components acting in trans on mRNAs are frequent causes of a large variety of different human disorders. The etiology of most of these diseases is, however, only poorly understood, mostly because the consequences for mRNA-metabolism are unclear. Here we discuss three prominent genetic diseases that fall into this category, namely spinal muscular atrophy (SMA), retinitis pigmentosa (RP) and X-linked syndromic mental retardation (XLMR). Whereas SMA and RP can be directly linked to mRNA processing, XLMR results from mutations in the mRNA surveillance system. We discuss how defects in mRNA maturation and turnover might lead to the tissue specific defects seen in these diseases.

Identification of a PRPF4 loss-of-function variant that abrogates U4/U6.U5 tri-snRNP integration and is associated with retinitis pigmentosa.

Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina.

Deletion of TOP3ß, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.

Implicating particular genes in the generation of complex brain and behavior phenotypes requires multiple lines of evidence. The rarity of most high-impact genetic variants typically precludes the possibility of accruing statistical evidence that they are associated with a given trait. We found that the enrichment of a rare chromosome 22q11.22 deletion in a recently expanded Northern Finnish sub-isolate enabled the detection of association between TOP3B and both schizophrenia and cognitive impairment. Biochemical analysis of TOP3ß revealed that this topoisomerase was a component of cytosolic messenger ribonucleoproteins (mRNPs) and was catalytically active on RNA. The recruitment of TOP3ß to mRNPs was independent of RNA cis-elements and was coupled to the co-recruitment of FMRP, the disease gene product in fragile X mental retardation syndrome. Our results indicate a previously unknown role for TOP3ß in mRNA metabolism and suggest that it is involved in neurodevelopmental disorders.

Analysis of photoreceptor degeneration in the zebrafish Danio rerio.

Disturbances in the general mRNA metabolism have been recognized as a major defect in a growing number of hereditary human diseases. One prominent example of this disease group is Retinitis pigmentosa (RP), characterized by selective loss of photoreceptor cells. RP can be caused by dominant mutations in key factors of the pre-mRNA processing spliceosome. In these cases, the complex events leading to the RP phenotype can only insufficiently be analyzed in rodents or other model organisms due to the essential functions of these splice factors. Here we introduce the zebrafish Danio rerio as a valuable vertebrate model system to study RP and related diseases.

The 1D4 antibody labels outer segments of long double cone but not rod photoreceptors in zebrafish.

PURPOSE: In experimental eye research, zebrafish has become a powerful model for human retina disorders. The purpose of the present study is the characterization of antibodies commonly employed in zebrafish models for rod photoreceptor degeneration. METHODS: The 1D4 monoclonal antibody, developed against bovine rhodopsin, has been widely used in studies addressing structural and functional features of rhodopsin and was reported as an informative marker to stain rod outer segments in both mice and zebrafish. We have used transgenic reporter lines and histologic analysis to determine the photoreceptor types identified by 1D4 and other antibodies in zebrafish. RESULTS: We demonstrate that 1D4, in contrast to what has been reported previously, does not recognize rod outer segments in zebrafish, but instead labels long double cone outer segments consistent with sequence conservation of the respective epitope. As an alternative marker for zebrafish rods, we characterized the monoclonal antibody zpr-3, which was found to stain outer segments of both rods, as well as double cones. CONCLUSIONS: Our findings highlight the importance to confirm specificity of antibodies in cross-species experiments for correct interpretation of experimental data. Our findings clarify conflicting published information arising from studies using 1D4 and zpr-3 antibodies in zebrafish.

Intronic miR-26b controls neuronal differentiation by repressing its host transcript, ctdsp2.

Differentiation of neural stem cells (NSCs) to neurons requires the activation of genes controlled by the repressor element 1 (RE1) silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF) protein complex. Important components of REST/NRSF are phosphatases (termed RNA polymerase II C-terminal domain small phosphatases [CTDSPs]) that inhibit RNA polymerase II and suppress neuronal gene expression in NSCs. Activation of genes controlled by CTDSPs is required for neurogenesis, but how this is achieved is not fully understood. Here we show that ctdsp2 is a target of miR-26b, a microRNA that is encoded in an intron of the ctdsp2 primary transcript. This intrinsic negative feedback loop is inactive in NSCs because miR-26b biogenesis is inhibited at the precursor level. Generation of mature miR-26b is activated during neurogenesis, where it suppresses Ctdsp2 protein expression and is required for neuronal cell differentiation in vivo.

Systemic splicing factor deficiency causes tissue-specific defects: a zebrafish model for retinitis pigmentosa.

Retinitis pigmentosa (RP) is a common hereditary eye disease that causes blindness due to a progressive loss of photoreceptors in the retina. RP can be elicited by mutations that affect the tri-snRNP subunit of the pre-mRNA splicing machinery, but how defects in this essential macromolecular complex transform into a photoreceptor-specific phenotype is unknown. We have modeled the disease in zebrafish by silencing the RP-associated splicing factor Prpf31 and observed detrimental effects on visual function and photoreceptor morphology. Despite reducing the level of a constitutive splicing factor, no general defects in gene expression were found. Instead, retinal genes were selectively affected, providing the first in vivo link between mutations in splicing factors and the RP phenotype. Silencing of Prpf4, a splicing factor hitherto unrelated to RP, evoked the same defects in vision, photoreceptor morphology and retinal gene expression. Hence, various routes affecting the tri-snRNP can elicit tissue-specific gene expression defects and lead to the RP phenotype.

Tdrd3 is a novel stress granule-associated protein interacting with the Fragile-X syndrome protein FMRP.

Tudor domains are widespread among proteins involved in RNA metabolism, but only in a few cases their cellular function has been analyzed in detail. Here, we report on the characterization of the ubiquitously expressed Tudor domain containing protein Tdrd3. Apart from its Tudor domain, we show that Tdrd3 possesses an oligosaccharide/nucleotide binding fold (OB-fold) and an ubiquitin associated domain capable of binding tetra-ubiquitin. A set of biochemical experiments revealed an interaction of Tdrd3 with FMRP, the product of the gene affected in Fragile X syndrome, and its autosomal homologs FXR1 and FXR2. FMRP has been implicated in the translational regulation of target mRNAs and shown to be a component of stress granules (SG). We demonstrate that overexpression of Tdrd3 in cells induces the formation of SGs and as a result leads to its co-localization with endogenous FMRP in these structures. Interestingly, the disease-associated FMRP missense mutation I304N identified in a Fragile X patient severely impairs the interaction with Tdrd3 in biochemical experiments. We propose a contribution of Tdrd3 to FMRP-mediated translational repression and suggest that the loss of the FMRP-Tdrd3 interaction caused by the I304N mutation might contribute to the pathogenesis of Fragile X syndrome.