MicroRNAs (miRNAs) are widespread among eukaryotes, and studies in several systems have revealed that miRNAs can regulate expression of specific genes. Primary miRNA transcripts are initially processed to ≈70-nucleotide (nt) stem–loop structures (pre-miRNAs), exported to the cytoplasm, further processed to yield ≈22-nt dsRNAs, and finally incorporated into ribonucleoprotein particles, which are thought to be the active species. Here we study nuclear export of pre-miRNAs and show that the process is saturable and thus carrier-mediated. Export is sensitive to depletion of nuclear RanGTP and, according to this criterion, mediated by a RanGTP-dependent exportin. An unbiased affinity chromatography approach with immobilized pre-miRNAs identified exportin 5 as the pre-miRNA-specific export carrier. We have cloned exportin 5 from Xenopus and demonstrate that antibodies raised against the Xenopus receptor specifically block pre-miRNA export from nuclei of Xenopus oocytes. We further show that exportin 5 interacts with double-stranded RNA in a sequence-independent manner.

RRNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilize the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase center and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2′-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity. While the mechanisms regulating partial modification and the functions of specialized ribosomes are largely unknown, changes in the rRNA modification pattern have been observed in response to environmental changes, during development, and in disease. This suggests that rRNA modifications may contribute to the translational control of gene expression.

N6-methyladenosine (m⁶A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m⁶A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m⁶A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m⁶A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5–TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m⁶A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5–TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.

5-methylcytosine (m⁵C) is an abundant RNA modification that’s presence is reported in a wide variety of RNA species, including cytoplasmic and mitochondrial ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), as well as messenger RNAs (mRNAs), enhancer RNAs (eRNAs) and a number of non-coding RNAs. In eukaryotes, C5 methylation of RNA cytosines is catalyzed by enzymes of the NOL1/NOP2/SUN domain (NSUN) family, as well as the DNA methyltransferase homologue DNMT2. In recent years, substrate RNAs and modification target nucleotides for each of these methyltransferases have been identified, and structural and biochemical analyses have provided the first insights into how each of these enzymes achieves target specificity. Functional characterizations of these proteins and the modifications they install have revealed important roles in diverse aspects of both mitochondrial and nuclear gene expression. Importantly, this knowledge has enabled a better understanding of the molecular basis of a number of diseases caused by mutations in the genes encoding m⁵C methyltransferases or changes in the expression level of these enzymes.