• Article
    | Open Access

    Current high-throughput single-cell transcriptomic methods are incompatible with paraformaldehyde, a common cell fixation technique. Here the authors present FD-seq, a method for droplet-based RNA sequencing of paraformaldehyde-fixed, stained and sorted single cells.

    • Hoang Van Phan
    • , Michiel van Gent
    •  & Savaş Tay
  • Article
    | Open Access

    C. elegans piRNA biogenesis and chromosome segregation (PICS) complex is composed of TOFU-6, PICS-1, ERH-2, and two mutually exclusive factors PID-1 and TOST-1. By employing biochemical, structural, and cellular biology methods, the authors show that the PICS complex is an octamer consisting of two copies of each subunit, and functions in piRNA biogenesis and mitosis.

    • Xiaoyang Wang
    • , Chenming Zeng
    •  & Chao Xu
  • Article
    | Open Access

    Met1-linked linear ubiquitination (M1-Ubi) is catalyzed by linear ubiquitin chain assembly complex (LUBAC). Here the authors show that Ago2 protein is M1-Ubi modified by LUBAC complex under hypoxia condition leading to less association of miRNA target mRNAs to Ago2 protein and de-repression of miRNA targets.

    • Hailong Zhang
    • , Xian Zhao
    •  & Jianxiu Yu
  • Article
    | Open Access

    m6A RNA post-transcriptional modification changes RNA hybridization kinetics. Here the authors show that the methylamino group can adopt syn-conformation pairing with uridine with a mismatch-like conformation in RNA duplex. They also develop a quantitative model that predicts how m6A affects the kinetics of hybridization.

    • Bei Liu
    • , Honglue Shi
    •  & Hashim M. Al-Hashimi
  • Article
    | Open Access

    Ribosome profiling has become the gold standard to analyze mRNA translation dynamics, and the translation inhibitor cycloheximide (CHX) is often used in its application. Here the authors systematically demonstrate that CHX does not bias the outcome of ribosome profiling experiments in most organisms.

    • Puneet Sharma
    • , Jie Wu
    •  & Sebastian A. Leidel
  • Article
    | Open Access

    Proximity labeling is used to map and discover proteins in specific subcellular compartments. Here the authors combine APEX-mediated proximity labeling with organic-aqueous phase separation to identify nuclear, nucleolar, and outer mitochondrial membrane RNA binding proteins.

    • Wei Qin
    • , Samuel A. Myers
    •  & Alice Y. Ting
  • Article
    | Open Access

    RNA polyadenosine tails are important for the export, translation and stability of mRNAs and play a role in non-coding RNA biogenesis. Here the authors measure yeast poly(A) tail lengths by direct RNA sequencing, revealing its dynamics in yeast exonuclease, deadenylase and poly(A) polymerase mutants.

    • Agnieszka Tudek
    • , Paweł S. Krawczyk
    •  & Andrzej Dziembowski
  • Article
    | Open Access

    Trans-translation, mediated by small protein B (SmpB) and transfer-messenger RNA (tmRNA), enables recycling of the ribosomes stalled on defective mRNAs in bacteria. Here, the authors report structures of the ribosome during trans-translation that reveal a translocation intermediate and elucidate the movements of the tmRNA-SmpB complex in the ribosome.

    • Charlotte Guyomar
    • , Gaetano D’Urso
    •  & Reynald Gillet
  • Article
    | Open Access

    Translational regulation by riboswitches is an important mechanism for the modulation of gene expression in bacteria. Here the authors show that the ligand-induced allosteric switch in the adenine-sensing riboswitch from V. vulnificus is insufficient and leads only to a partial opening of the ribosome binding site and requires interaction with 30S-bound ribosomal protein S1, which acts as an RNA chaperone.

    • Vanessa de Jesus
    • , Nusrat S. Qureshi
    •  & Boris Fürtig
  • Article
    | Open Access

    Ribosome biogenesis is crucially dependent on proper rRNA folding, a process assisted by chaperones. Here the authors reveal how Puf6 promotes correct rRNA folding at low temperature, a condition where mis-paired RNA folding intermediates frequently accumulate.

    • Stefan Gerhardy
    • , Michaela Oborská-Oplová
    •  & Vikram Govind Panse
  • Article
    | Open Access

    Assembly of the mitoribosome requires assistance from numerous specialized factors. Here, structures of the human 39S late assembly intermediates identify several assembly factors which keep the 16S rRNA in immature conformations, and reveal deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly.

    • Jingdong Cheng
    • , Otto Berninghausen
    •  & Roland Beckmann
  • Article
    | Open Access

    Fly Dicer-2 is thought to use two distinct – processive or distributive – modes of cleavage by distinguishing the terminal structures of double-stranded RNA (dsRNA) substrates with the help of its cofactor LoquaciousPD (Loqs-PD). Here the authors show by single-molecule imaging that dsRNA terminal structures and Loqs-PD change the probability for Dicer to initiate processive cleavage but not the mode of cleavage action per se.

    • Masahiro Naganuma
    • , Hisashi Tadakuma
    •  & Yukihide Tomari
  • Article
    | Open Access

    RNA modifications appear to play a role in determining RNA structure and function. Here, the authors develop a deep learning model that predicts the location of 12 RNA modifications using primary sequence, and show that several modifications are associated, which suggests dependencies between them.

    • Zitao Song
    • , Daiyun Huang
    •  & Jia Meng
  • Article
    | Open Access

    In humans, protein methyltransferase is responsible for RNA methylation using S-adenosylmethionine as a methyl group donor. Here the authors report a self-methylation activity of a bacterial riboswitch.

    • Laurin Flemmich
    • , Sarah Heel
    •  & Ronald Micura
  • Article
    | Open Access

    Mitochondrial ribosomes (mitoribosomes) are characterized by a distinct architecture and thus biogenesis pathway. Here, cryo-EM structures of mitoribosome large subunit assembly intermediates elucidate final steps of 16 S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, as well as functions of several mitoribosome assembly factors.

    • Miriam Cipullo
    • , Genís Valentín Gesé
    •  & Joanna Rorbach
  • Article
    | Open Access

    Mitochondrial ribosomes (mitoribosomes) are characterized by a distinct architecture and thus biogenesis pathway. Here, cryo-EM structures of mitoribosome large subunit assembly intermediates elucidate final steps of 16 S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, as well as functions of several mitoribosome assembly factors.

    • Caillan Crowe-McAuliffe
    • , Victoriia Murina
    •  & Daniel N. Wilson
  • Article
    | Open Access

    Fluorogenic RNA aptamers such as Chili display strong fluorescence enhancement upon aptamer–ligand complex formation. Here, the authors provide insights into the mechanism of fluorescence activation of Chili by solving the crystal structures of Chili with its bound positively charged ligands DMHBO+ and DMHBI+, and they reveal that Chili uses an excited state proton transfer mechanism based on time-resolved optical spectroscopy measurements.

    • Mateusz Mieczkowski
    • , Christian Steinmetzger
    •  & Claudia Höbartner
  • Article
    | Open Access

    Sequencing methods such as icSHAPE were developed to probe RNA structures transcriptome-wide in cells. To probe intact RNA structures, the authors develop icSHAPE-MaP and apply to Dicer-bound substrates showing that distance measuring is important for Dicer cleavage of pre-miRNAs.

    • Qing-Jun Luo
    • , Jinsong Zhang
    •  & Qiangfeng Cliff Zhang
  • Article
    | Open Access

    Long non coding RNA TERRA transcripts can form R-loops at chromosome ends. Here, the authors reveal a role for the helicase RTEL in affecting TERRA levels and localization.

    • Fiorella Ghisays
    • , Aitor Garzia
    •  & John H. J. Petrini
  • Article
    | Open Access

    Macrolide antibiotics inhibit bacterial translation in a context-specific manner, arresting ribosomes at defined sites within mRNAs and selectively inhibiting synthesis of only a subset of cellular proteins. Here the authors provide a structural basis for the context-specific activity of macrolides on the eukaryotic ribosome.

    • Maxim S. Svetlov
    • , Timm O. Koller
    •  & Alexander S. Mankin
  • Article
    | Open Access

    In some systems, a single protein comprising reverse transcriptase (RT), integrase and maturase enables concerted sequence integration and crRNA production. Here, analyses including the structure of a Cas6-RT-Cas1—Cas2 complex suggest coordination between all three active sites and capacity to acquire CRISPR sequences from RNA and DNA substrates.

    • Joy Y. Wang
    • , Christopher M. Hoel
    •  & Jennifer A. Doudna
  • Article
    | Open Access

    Flaviviruses use a ~70 nucleotide stem-loop structure called stem-loop A (SLA) at the 5’ end of the RNA genome as a promoter for RNA synthesis by the viral polymerase NS5. Here the authors describe the structures of dengue and Zika virus SLAs, identify the SLA-binding site on NS5, and propose models for how NS5 recognizes the RNA promoter.

    • Eunhye Lee
    • , Paul J. Bujalowski
    •  & Kyung H. Choi
  • Article
    | Open Access

    Initiation of HIV-1 reverse transcription occurs at the host tRNALys3, which forms a complex with the 5’ end of the HIV-1 viral RNA and reverse transcriptase (RT). Here, the authors present the 2.8 Å cryo-EM structure of a minimal HIV-1 RT–vRNA–tRNALys3 initiation complex (miniRTIC), and miniRTIC structures with the bound non-nucleoside reverse transcriptase inhibitors nevirapine and efavirenz at 3.1 and 2.9 Å resolution, respectively.

    • Betty Ha
    • , Kevin P. Larsen
    •  & Elisabetta Viani Puglisi
  • Article
    | Open Access

    RNA crosslinking and proximity ligation methods are used to identify transcriptome-wide base pairing interactions. Here, the authors report PARIS2 (psoralen analysis of RNA interactions and structures 2), a method for RNA duplex determination in vivo with higher efficiency than the previous PARIS method.

    • Minjie Zhang
    • , Kongpan Li
    •  & Zhipeng Lu
  • Article
    | Open Access

    Time-resolved crystallography (TRX) is used for monitoring only small conformational changes of biomacromolecules within the same lattice. Here, the authors report the interplay between synchronous molecular rearrangements and lattice phase transitions in RNA crystals, providing the basis for the investigation of large conformational changes using TRX.

    • Saminathan Ramakrishnan
    • , Jason R. Stagno
    •  & Yun-Xing Wang
  • Article
    | Open Access

    The demethylase FTO was shown to remove on N6-methyladenosine (m6A) and N6, 2’-O-dimethyladenosine (m6Am) modifications on RNAs. Here the authors show that FTO impedes cancer stem cell-like abilities in colorectal cancer cells through its m6Am demethylase activity, not through internal m6A demethylase activity.

    • Sébastien Relier
    • , Julie Ripoll
    •  & Alexandre David
  • Article
    | Open Access

    ATP-dependent helicases remodel the spliceosome and proofread splice site recognition. A new method – Purified Spliceosome iCLIP (psiCLIP) – probes protein-RNA interactions in defined spliceosome complexes to reveal how the helicases Prp16 and Prp22 promote correct mRNA synthesis through dynamic binding on their RNA substrates.

    • Lisa M. Strittmatter
    • , Charlotte Capitanchik
    •  & Kiyoshi Nagai
  • Article
    | Open Access

    The noncoding RNA 7SK controls the transcription of mRNAs. Here, the authors show that the 7SK complex interacts with the Smn complex, suggesting crosstalk between transcription and snRNP assembly.

    • Changhe Ji
    • , Jakob Bader
    •  & Michael Briese
  • Article
    | Open Access

    Nematode P granules are cytoplasmic RNA–protein biomolecule condensates central to germ cell development. Here the authors show that dimerization of the PGL-1 scaffolding protein is crucial to granule formation and mRNA repression, and that the WAGO-1 Argonaute protein is a cofactor in repressing PGL-1 bound mRNAs.

    • Scott Takeo Aoki
    • , Tina R. Lynch
    •  & Judith Kimble
  • Article
    | Open Access

    Accurately predicting the secondary structure of non-coding RNAs can help unravel their function. Here the authors propose a method integrating thermodynamic information and deep learning to improve the robustness of RNA secondary structure prediction compared to several existing algorithms.

    • Kengo Sato
    • , Manato Akiyama
    •  & Yasubumi Sakakibara
  • Article
    | Open Access

    The SARS-CoV-2 nucleocapsid (N) protein binds the viral RNA genome and contains two ordered domains flanked by three intrinsically-disordered regions. Here, the authors show that RNA binding induces liquid-liquid phase separation of N, which is driven by its central intrinsically-disordered region and is modulated by phosphorylation. The SARS-CoV-2 Membrane (M) protein also phase-separates with N, and three-component mixtures of N + M + RNA form mutually exclusive compartments containing N + M or N + RNA.

    • Shan Lu
    • , Qiaozhen Ye
    •  & Kevin D. Corbett
  • Article
    | Open Access

    SRSF1 is an oncoprotein that plays important roles in RNA metabolism. We reveal the structure of the human SRSF1 RRM1 bound to RNA, and propose a bimodal mode of interaction of the protein with RNA. A single mutation in RRM1 changed SRSF1 specificity for RNA and made it active on SMN2 exon7 splicing.

    • Antoine Cléry
    • , Miroslav Krepl
    •  & Frédéric H.-T. Allain
  • Article
    | Open Access

    Post transcriptional modification of RNAs represents an important layer of gene regulation. Here the authors describe NAIL-MS—a method for monoisotopic RNA labeling in cell culture—demonstrating its capabilities by analyzing the modification kinetics of total tRNA, 18S rRNA and tRNAPhe as models.

    • Matthias Heiss
    • , Felix Hagelskamp
    •  & Stefanie Kellner
  • Article
    | Open Access

    H2A.X histone variant gene encodes poly(A)+ and poly(A)- mRNA isoforms which are differentially expressed depending on cell lines. Here the authors show that upon DNA damage, cells expressing more poly(A)+ isoform require this isoform for de novo H2A.X synthesis while cells with more poly(A)- isoform have sufficient H2A.X present in chromatin.

    • Esther Griesbach
    • , Margarita Schlackow
    •  & Nick J. Proudfoot
  • Article
    | Open Access

    Remdesivir is a nucleoside analog that inhibits the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and is used as a drug to treat COVID19 patients. Here, the authors provide insights into the mechanism of remdesivir-induced RdRp stalling by determining the cryo-EM structures of SARS-CoV-2 RdRp with bound RNA molecules that contain remdesivir at defined positions and observe that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation.

    • Goran Kokic
    • , Hauke S. Hillen
    •  & Patrick Cramer
  • Article
    | Open Access

    Circular RNAs (circRNA) is a class of non-coding RNAs that can regulate gene translation and function. Here the authors show that a circRNA, circNDUFB2, is downregulated in non-small cell lung cancer tissues, and likely contributes to anti-tumor immunity by regulating both degradation of oncoproteins and induction of innate immunity.

    • Botai Li
    • , Lili Zhu
    •  & Wenxin Qin