Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1, which regulates a variety of physiological processes including cancer progression and corpus luteum formation. To obtain further insight into the molecular basis of the function of paraspeckles, we performed fine structural analyses of these nuclear bodies using structural illumination microscopy. Notably, paraspeckle proteins are found within different layers along the radially arranged bundles of Neat1 transcripts, forming a characteristic core-shell spheroidal structure. In cells lacking the RNA binding protein Fus, paraspeckle spheroids are disassembled into smaller particles containing Neat1, which are diffusely distributed in the nucleoplasm. Sequencing analysis of RNAs purified from paraspeckles revealed that AG-rich transcripts associate with Neat1, which are distributed along the shell of the paraspeckle spheroids. We propose that paraspeckles sequester core components inside the spheroids, whereas the outer surface associates with other components in the nucleoplasm to fulfill their function.

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m¹A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m¹A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m¹A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function. RNA modifications constitute an important layer of information, with functional implications that are not written in the underlying DNA sequence. Recently, we observed an apparent RNA-DNA difference (RDD) at position 947 of the human mitochondrial 16S ribosomal RNA (rRNA), but its nature and mechanism were unclear. Here we show that this disparity reflects an m¹A modification (methylation at position 1 of the adenine moiety), and demonstrated by a combination of knock-down experiments in cells and in vitro methylation assays that the tRNA methyltransferase TRMT61B is the best candidate enzyme to introduce this modification. We also show that this modification is present in most of the 16S rRNA molecules in isolated mitochondrial ribosomes, and that it occurs in all vertebrates with an adenine (90% of the vertebrates), but not in those with a thymidine at this 16S rRNA position. Finally, as the first step towards understanding the functional importance of this rRNA modification, we used a genome-edited bacterial system to demonstrate that an unmodified adenine reduced the growth and translation rates of the bacteria as compared to both wild-type bacteria and mutant bacteria with a thymidine in the relevant position. Hence, three solutions were selected during evolution to allow proper function of the mitochondrial 16S rRNA—either RNA modification or two alternative ancient evolutionary DNA mutations.

Defective tRNA 2′- O -modification impairs translation efficiency at Phe codon in the brain and induces a neurological dysfunction.

Naked mole-rats (NMRs) have a very low spontaneous carcinogenesis rate, which has prompted studies on the responsible mechanisms to provide clues for human cancer prevention. However, it remains unknown whether and how NMR tissues respond to experimental carcinogenesis induction. Here, we show that NMRs exhibit extraordinary resistance against potent chemical carcinogenesis induction through a dampened inflammatory response. Although carcinogenic insults damaged skin cells of both NMRs and mice, NMR skin showed markedly lower immune cell infiltration. NMRs harbour loss-of-function mutations in RIPK3 and MLKL genes, which are essential for necroptosis, a type of necrotic cell death that activates strong inflammation. In mice, disruption of Ripk3 reduced immune cell infiltration and delayed carcinogenesis. Therefore, necroptosis deficiency may serve as a cancer resistance mechanism via attenuating the inflammatory response in NMRs. Our study sheds light on the importance of a dampened inflammatory response as a non-cell-autonomous cancer resistance mechanism in NMRs.

In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f⁵C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3^HKO) mice. Nsun3^HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3^HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3^HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian embryonic development and shows that tissue-specific loss of a single mitochondrial tRNA modification can induce tissue aberration that worsens in later adulthood.

Retroviral infection requires reverse transcription, and the reverse transcriptase (RT) uses cellular tRNA as its primer. In humans, the TRMT6-TRMT61A methyltransferase complex incorporates N¹-methyladenosine modification at tRNA position 58 (m¹A58); however, the role of m¹A58 as an RT-stop site during retroviral infection has remained questionable. Here, we constructed TRMT6 mutant cells to determine the roles of m¹A in HIV-1 infection. We confirmed that tRNA₃^Lys m¹A58 was required for in vitro plus-strand strong-stop by RT. Accordingly, infectivity of VSV-G pseudotyped HIV-1 decreased when the virus contained m¹A58-deficient tRNA₃^Lys instead of m¹A58-modified tRNA₃^Lys. In TRMT6 mutant cells, the global protein synthesis rate was equivalent to that of wild-type cells. However, unexpectedly, plasmid-derived HIV-1 expression showed that TRMT6 mutant cells decreased accumulation of HIV-1 capsid, integrase, Tat, Gag, and GagPol proteins without reduction of HIV-1 RNAs in cells, and fewer viruses were produced. Moreover, the importance of 5,2′-O-dimethyluridine at U54 of tRNA₃^Lys as a second RT-stop site was supported by conservation of retroviral genome-tRNA^Lys sequence-complementarity, and TRMT6 was required for efficient 5-methylation of U54. These findings illuminate the fundamental importance of tRNA m¹A58 modification in both the early and late steps of HIV-1 replication, as well as in the cellular tRNA modification network.