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In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.

Nucleic acids research : NAR 49(16), e92 (2021). doi:10.1093/nar/gkab485, Published by Oxford Univ. Press, Oxford

Phylogenetic analyses of nuclear and mitochondrial genomes indicate that polar bears captured the brown bear mitochondrial genome 160,000 years ago, leading to an extinction of the original polar bear mitochondrial genome. However, mitochondrial DNA occasionally integrates into the nuclear genome, forming pseudogenes called numts (nuclear mitochondrial integrations). Screening the polar bear genome identified only 13 numts. Genomic analyses of two additional ursine bears and giant panda indicate that all except one of the discovered numts entered the bear lineage at least 14 million years ago. However, short read genome assemblies might lead to an under-representation of numts or other repetitive sequences. Our findings suggest low integration rates of numts in bears and a loss of the original polar bear mitochondrial genome.

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimised miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses are calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.HighlightsmiCLIP2 produces complex libraries to map m6A RNA modificationsMettl3 KO miCLIP2 allows to identify Mettl3-dependent RNA modification sitesMachine learning predicts genuine m6A sites from human and mouse miCLIP2 data without Mettl3 KOm6A modifications occur outside of DRACH motifs and associate with alternative splicing

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3’ UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells we show that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. We observe increased association of the translational repressor Bru1 with osk mRNA upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3’ UTR., Author summary To ensure accurate development of the Drosophila embryo, proteins and mRNAs are positioned at specific sites within the embryo. Many of these factors are produced and localized during the development of the egg in the mother. One protein essential for this process that has been heavily studied is Oskar (Osk), which is positioned at the posterior pole. During the localization of osk mRNA, its translation is repressed by the RNA-binding protein Bruno1 (Bru1), ensuring that Osk protein is not present outside of the posterior where it is harmful. At the posterior pole, osk mRNA is activated through mechanisms that are not yet understood. In this work, we show that the conserved protein Makorin 1 (Mkrn1) is a novel factor involved in the translational activation of osk. Mkrn1 binds specifically to osk mRNA, overlapping with a binding site of Bru1, thus alleviating the association of Bru1 with osk. Moreover, Mkrn1 is stabilized by poly(A) binding protein (pAbp), a translational activator that binds osk mRNA in close proximity to one Mkrn1 binding site. Our work thus helps to answer a long-standing question in the field, providing insight about the function of Mkrn1 and more generally into embryonic patterning in animals.

Cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via the ribosome-associated quality control (RQC). Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during RQC. We show that MKRN1 interacts with the cytoplasmic poly(A)-binding protein (PABP) and is positioned upstream of poly(A) tails in mRNAs. Ubiquitin remnant profiling uncovers PABP and ribosomal protein RPS10, as well as additional translational regulators as main ubiquitylation substrates of MKRN1. We propose that MKRN1 serves as a first line of poly(A) recognition at the mRNA level to prevent production of erroneous proteins, thus maintaining proteome integrity.

Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. InDrosophilamaternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation ofoskar. We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and bindsosk3’ UTR in a region adjacent to A-rich sequences. This binding site overlaps with Bruno1 (Bru1) responsive elements (BREs), which regulateosktranslation. We observe increased association of the translational repressor Bru1 withoskmRNA upon depletion of Mkrn1, indicating that both proteins compete foroskbinding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries fromMkrn1females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activatingosktranslation by displacing Bru1 from its 3’ UTR.Author SummaryTo ensure accurate development of theDrosophilaembryo, proteins and mRNAs are positioned at specific sites within the embryo. Many of these proteins and mRNAs are produced and localized during the development of the egg in the mother. One protein essential for this process that has been heavily studied is Oskar (Osk), which is positioned at the posterior pole. During the localization ofoskmRNA, its translation is repressed by the RNA-binding protein Bruno1 (Bru1), ensuring that Osk protein is not present outside of the posterior where it is harmful. At the posterior pole,oskmRNA is activated through mechanisms that are not yet understood. In this work, we show that the conserved protein Makorin 1 (Mkrn1) is a novel factor involved in the translational activation ofosk. Mkrn1 binds specifically tooskmRNA in a region that overlaps with the binding site of Bru1, thus alleviating the association of Bru1 withosk. Moreover, Mkrn1 is stabilized by poly(A) binding protein, a translational activator that bindsoskmRNA in close proximity to Mkrn1. Our work thus helps to answer a long-standing question in the field, providing insight about the function of Mkrn1 and more generally into embryonic patterning in animals.

Phylogenetic analyses of nuclear and mitochondrial genomes have shown that polar bears captured the mitochondrial genome of brown bears some 160,00 years ago. This hybridization event likely led to an extinction of the original polar bear mitochondrial genome. However, parts of the mitochondrial DNA occasionally integrates into the nuclear genome, forming pseudogenes called numts (nuclear mitochondrial integrations). Screening the polar bear genome for numts, we identified only 13 such integrations. Analyses of whole-genome sequences from additional polar bears, brown and American black bears as well as the giant panda indicates that the discovered numts entered the bear lineage before the initial ursid radiation some 14 million years ago. Our findings suggests a low integration rate of numts in the bear lineage and a complete loss of the original polar bear mitochondrial genome.