The steady-state levels of microRNAs (miRNAs) and their activities are regulated by the post-transcriptional processes. It is known that 39 ends of several miRNAs undergo post-dicing adenylation or uridylation. We isolated the liver-specific miR-122 from human hepatocytes and mouse livers. Direct analysis by mass spectrometry revealed that one variant of miR-122 has a 39-terminal adenosine that is introduced after processing by Dicer. We identified GLD-2, which is a regulatory cytoplasmic poly(A) polymerase, as responsible for the 39-terminal adenylation of miR-122 after unwinding of the miR-122/ miR-122* duplex. In livers from GLD-2-null mice, the steady-state level of the mature form of miR-122 was specifically lower than in heterozygous mice, whereas no reduction of pre-miR-122 was observed, demonstrating that 39-terminal adenylation by GLD-2 is required for the selective stabilization of miR-122 in the liver.Supplemental material is available at http://www.genesdev.org.

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway.

Piwi-interactingSupplemental material is available at http://www.genesdev.org.

The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm5s2U by largely fixing the C3′-endo ribose puckering, ensuring stable and accurate codon–anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm5s2U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

Summary PIWI-interacting RNAs (piRNAs) play a crucial role in transposon silencing in animal germ cells. In piRNA biogenesis, single-stranded piRNA intermediates are loaded into PIWI-clade proteins and cleaved by Zucchini/MitoPLD, yielding precursor piRNAs (pre-piRNAs). Pre-piRNAs that are longer than the mature piRNA length are then trimmed at their 3′ ends. Although recent studies implicated the Tudor domain protein Papi/Tdrkh in pre-piRNA trimming, the identity of Trimmer and its relationship with Papi/Tdrkh remain unknown. Here, we identified PNLDC1, an uncharacterized 3′–5′ exonuclease, as Trimmer in silkworms. Trimmer is enriched in the mitochondrial fraction and binds to Papi/Tdrkh. Depletion of Trimmer and Papi/Tdrkh additively inhibits trimming, causing accumulation of ~35–40-nt pre-piRNAs that are impaired for target cleavage and prone to degradation. Our results highlight the cooperative action of Trimmer and Papi/Tdrkh in piRNA maturation.

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of posttranscriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.

In human mitochondria, 10 mRNAs species are generated from a long polycistronic precursor that is transcribed from the heavy chain of mitochondrial DNA, in theory yielding equal copy numbers of mRNA molecules. However, the steady-state levels of these mRNAs differ substantially. Through absolute quantification of mRNAs in HeLa cells, we show that the copy numbers of all mitochondrial mRNA species range from 6000 to 51 000 molecules per cell, indicating that mitochondria actively regulate mRNA metabolism. In addition, the copy numbers of mitochondrial mRNAs correlated with their cellular half-life. Previously, mRNAs with longer half-lives were shown to be stabilized by the LRPPRC/SLIRP complex, which we find that cotranscriptionally binds to coding sequences of mRNAs. We observed that the LRPPRC/SLIRP complex suppressed 3′ exonucleolytic mRNA degradation mediated by PNPase and SUV3. Moreover, LRPPRC promoted the polyadenylation of mRNAs mediated by mitochondrial poly(A) polymerase (MTPAP) in vitro. These findings provide a framework for understanding the molecular mechanism of mRNA metabolism in human mitochondria.

Piwi-interacting RNAs (piRNAs) are a germline-specific class of small noncoding RNAs that are essential for spermatogenesis, but their function and biogenesis remain elusive. Here we report a post-transcriptional modification of mouse piRNAs. Mass spectrometric analysis reveals that the piRNAs tested are fully modified by 2'-O-methylation at their 3' termini. This observation may provide a clue to the biogenesis and function of piRNAs in spermatogenesis.