Dual modes of ZFC3H1 confer selectivity in nuclear RNA sorting.

The export and degradation pathways compete to sort nuclear RNAs, yet the default pathway remains unclear. Sorting of mature RNAs to degradation, facilitated by the exosome co-factor poly(A) exosome targeting (PAXT), is particularly challenging for their resemblance to mRNAs intended for translation. Here, we unveil that ZFC3H1, a core PAXT component, is co-transcriptionally loaded onto the first exon/intron of RNA precursors (pre-RNAs). Interestingly, this initial loading does not lead to pre-RNA degradation, as ZFC3H1 adopts a "closed" conformation, effectively blocking exosome recruitment. As processing progresses, RNA fate can be reshaped. Longer RNAs with more exons are allowed for nuclear export. By contrast, short RNAs with fewer exons preferentially recruit transient PAXT components ZC3H3 and RBM26/27 to the 3' end, triggering ZFC3H1 "opening" and subsequent exosomal degradation. Together, the decoupled loading and activation of ZFC3H1 pre-configures RNA fate for decay while still allowing a switch to nuclear export, depending on mature RNA features.

Technological advancements in deciphering RNA-RNA interactions.

RNA-RNA interactions (RRIs) can dictate RNA molecules to form intricate higher-order structures and bind their RNA substrates in diverse biological processes. To elucidate the function, binding specificity, and regulatory mechanisms of various RNA molecules, especially the vast repertoire of non-coding RNAs, advanced technologies and methods that globally map RRIs are extremely valuable. In the past decades, many state-of-the-art technologies have been developed for this purpose. This review focuses on those high-throughput technologies for the global mapping of RRIs. We summarize the key concepts and the pros and cons of different technologies. In addition, we highlight the novel biological insights uncovered by these RRI mapping methods and discuss the future challenges for appreciating the crucial roles of RRIs in gene regulation across bacteria, viruses, archaea, and mammals.

Protocol for profiling virus-to-host RNA-RNA interactions in infected cells by RIC-seq.

Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions remains challenging. Here, we present an RNA in situ conformation sequencing (RIC-seq)-based protocol for mapping high-confidence virus-to-host in situ RNA-RNA interactions in infected cells. We detail steps for formaldehyde crosslinking, pCp-biotin labeling, in situ proximity ligation, chimeric RNA enrichment, strand-specific library construction, and data analysis. This protocol allows unbiased identification of virus-to-host RNA-RNA interactions for various RNA viruses and is potentially applicable to DNA virus-derived transcripts. For complete details on the use and execution of this protocol, please refer to Zhao et al.1.

SARS-CoV-2 RNA stabilizes host mRNAs to elicit immunopathogenesis.

SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3' UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development.

Complementary Alu sequences mediate enhancer-promoter selectivity.

Enhancers determine spatiotemporal gene expression programs by engaging with long-range promoters1-4. However, it remains unknown how enhancers find their cognate promoters. We recently developed a RNA in situ conformation sequencing technology to identify enhancer-promoter connectivity using pairwise interacting enhancer RNAs and promoter-derived noncoding RNAs5,6. Here we apply this technology to generate high-confidence enhancer-promoter RNA interaction maps in six additional cell lines. Using these maps, we discover that 37.9% of the enhancer-promoter RNA interaction sites are overlapped with Alu sequences. These pairwise interacting Alu and non-Alu RNA sequences tend to be complementary and potentially form duplexes. Knockout of Alu elements compromises enhancer-promoter looping, whereas Alu insertion or CRISPR-dCasRx-mediated Alu tethering to unregulated promoter RNAs can create new loops to homologous enhancers. Mapping 535,404 noncoding risk variants back to the enhancer-promoter RNA interaction maps enabled us to construct variant-to-function maps for interpreting their molecular functions, including 15,318 deletions or insertions in 11,677 Alu elements that affect 6,497 protein-coding genes. We further demonstrate that polymorphic Alu insertion at the PTK2 enhancer can promote tumorigenesis. Our study uncovers a principle for determining enhancer-promoter pairing specificity and provides a framework to link noncoding risk variants to their molecular functions.

CRIC-seq protocol for in situ profiling of proximal RNA-RNA contacts associated with RNA-binding proteins.

RNA-binding proteins (RBPs) can bind and mediate RNA-RNA contacts. However, identifying specific RBP-organized RNA-RNA contacts remains challenging. Here, we present a capture RIC-seq (CRIC-seq) technique to map specific RBP-associated RNA-RNA contacts globally. We describe steps for formaldehyde cross-linking to fix RNA in situ conformation, pCp-biotin labeling to mark RNA juncture, and in situ proximity ligation to join proximal RNAs. We then detail immunoprecipitation to isolate specific RBP-associated RNA-RNA contacts, biotin-streptavidin selection to enrich chimeric RNAs, and library construction for paired-end sequencing. For complete information on the generation and use of this protocol, please refer to Ye et al.1.

Targeting intratumor heterogeneity suppresses colorectal cancer chemoresistance and metastasis.

Intratumor heterogeneity (ITH) is a barrier to effective therapy. However, it is largely unknown how ITH is established at the onset of tumor progression, such as in colorectal cancer (CRC). Here, we integrate single-cell RNA-seq and functional validation to show that asymmetric division of CRC stem-like cells (CCSC) is critical for early ITH establishment. We find that CCSC-derived xenografts contain seven cell subtypes, including CCSCs, that dynamically change during CRC xenograft progression. Furthermore, three of the subtypes are generated by asymmetric division of CCSCs. They are functionally distinct and appear at the early stage of xenografts. In particular, we identify a chemoresistant and an invasive subtype, and investigate the regulators that control their generation. Finally, we show that targeting the regulators influences cell subtype composition and CRC progression. Our findings demonstrate that asymmetric division of CCSCs contributes to the early establishment of ITH. Targeting asymmetric division may alter ITH and benefit CRC therapy.

RNA-binding protein complex AMG-1/SLRP-1 mediates germline development and spermatogenesis by maintaining mitochondrial homeostasis in Caenorhabditis elegans.

The mechanisms of RNA-binding proteins (RBPs)-mediated post-transcriptional regulation of pre-existing mRNAs, which is essential for spermatogenesis, remain poorly understood. In this study, we identify that a germline-specific mitochondrial RBP AMG-1(abnormal mitochondria in germline 1), a homolog of mammalian leucine-rich PPR motif-containing protein (LRPPRC), is required for spermatogenesis in Caenorhabditis elegans. The amg-1 mutation hinders germline development without affecting somatic development and leads to the aberrant mitochondrial morphology and structure associated with mitochondrial dysfunctions specifically in the germline. We demonstrate that AMG-1 is most frequently bound to mtDNA-encoded 12S and 16S ribosomal RNA, the essential components of mitochondrial ribosomes, and that 12S rRNA expression mediated by AMG-1 is crucial for germline mitochondrial protein homeostasis. Furthermore, steroid receptor RNA activator (SRA) stem loop interacting RNA binding protein (SLRP-1), a homolog of mammalian SRA stem loop interacting RNA binding protein (SLIRP) in C. elegans, interacts with AMG-1 genetically to regulate germline development and reproductive success in C. elegans. Overall, these findings reveal the novel function of mtRBP, specifically in regulating germline development.

NOVA1 prevents overactivation of the unfolded protein response and facilitates chromatin access during human white adipogenesis.

The molecular mechanism underlying white adipogenesis in humans has not been fully elucidated beyond the transcriptional level. Here, we found that the RNA-binding protein NOVA1 is required for the adipogenic differentiation of human mesenchymal stem cells. By thoroughly exploring the interactions between NOVA1 and its binding RNA, we proved that NOVA1 deficiency resulted in the aberrant splicing of DNAJC10 with an in-frame premature stop codon, reduced DNAJC10 expression at the protein level and hyperactivation of the unfolded protein response (UPR). Moreover, NOVA1 knockdown abrogated the down-regulation of NCOR2 during adipogenesis and up-regulated the 47b+ splicing isoform, which led to decreased chromatin accessibility at the loci of lipid metabolism genes. Interestingly, these effects on human adipogenesis could not be recapitulated in mice. Further analysis of multispecies genomes and transcriptomes indicated that NOVA1-targeted RNA splicing is evolutionarily regulated. Our findings provide evidence for human-specific roles of NOVA1 in coordinating splicing and cell organelle functions during white adipogenesis.

Sequencing of 19,219 exomes identifies a low-frequency variant in FKBP5 promoter predisposing to high myopia in a Han Chinese population.

High myopia (HM) is one of the leading causes of visual impairment and blindness worldwide. Here, we report a whole-exome sequencing (WES) study in 9,613 HM cases and 9,606 controls of Han Chinese ancestry to pinpoint HM-associated risk variants. Single-variant association analysis identified three newly identified -genetic loci associated with HM, including an East Asian ancestry-specific low-frequency variant (rs533280354) in FKBP5. Multi-ancestry meta-analysis with WES data of 2,696 HM cases and 7,186 controls of European ancestry from the UK Biobank discerned a newly identified European ancestry-specific rare variant in FOLH1. Functional experiments revealed a mechanism whereby a single G-to-A transition at rs533280354 disrupted the binding of transcription activator KLF15 to the promoter of FKBP5, resulting in decreased transcription of FKBP5. Furthermore, burden tests showed a significant excess of rare protein-truncating variants among HM cases involved in retinal blood vessel morphogenesis and neurotransmitter transport.

LSM14B is an Oocyte-Specific RNA-Binding Protein Indispensable for Maternal mRNA Metabolism and Oocyte Development in Mice.

Mammalian oogenesis features reliance on the mRNAs produced and stored during early growth phase. These are essential for producing an oocyte competent to undergo meiotic maturation and embryogenesis later when oocytes are transcriptionally silent. The fate of maternal mRNAs hence ensures the success of oogenesis and the quality of the resulting eggs. Nevertheless, how the fate of maternal mRNAs is determined remains largely elusive. RNA-binding proteins (RBPs) are crucial regulators of oogenesis, yet the identity of the full complement of RBPs expressed in oocytes is unknown. Here, a global view of oocyte-expressed RBPs is presented: mRNA-interactome capture identifies 1396 RBPs in mouse oocytes. An analysis of one of these RBPs, LSM family member 14 (LSM14B), demonstrates that this RBP is specific to oocytes and associated with many networks essential for oogenesis. Deletion of Lsm14b results in female-specific infertility and a phenotype characterized by oocytes incompetent to complete meiosis and early embryogenesis. LSM14B serves as an interaction hub for proteins and mRNAs throughout oocyte development and regulates translation of a subset of its bound mRNAs. Therefore, RNP complexes tethered by LSM14B are found exclusively in oocytes and are essential for the control of maternal mRNA fate and oocyte development.

Sex-lethal regulates back-splicing and generation of the sex-differentially expressed circular RNAs.

Conversely to canonical splicing, back-splicing connects the upstream 3' splice site (SS) with a downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely identified and have regulatory functions in eukaryotic gene expression. However, sex-specific back-splicing in Drosophila has not been investigated and its regulation remains unclear. Here, we performed multiple RNA analyses of a variety sex-specific Drosophila samples and identified over ten thousand circular RNAs, in which hundreds are sex-differentially and -specifically back-spliced. Intriguingly, we found that expression of SXL, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene that is only spliced into functional proteins in females, promoted back-splicing of many female-differential circRNAs in the male S2 cells, whereas expression of a SXL mutant (SXLRRM) did not promote those events. Using a monoclonal antibody, we further obtained the transcriptome-wide RNA-binding sites of SXL through PAR-CLIP. After splicing assay of mini-genes with mutations in the SXL-binding sites, we revealed that SXL-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing, whereas SXL-binding on the circRNA exons inhibits back-splicing. This study provides strong evidence that SXL has a regulatory role in back-splicing to generate sex-specific and -differential circRNAs, as well as in the initiation of sex-determination cascade through canonical forward-splicing.

Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export.

Skeletal muscle stem cells (also known as satellite cells [SCs]) are essential for muscle regeneration and the regenerative activities of SCs are intrinsically governed by gene regulatory mechanisms, but the post-transcriptional regulation in SCs remains largely unknown. N(6)-methyladenosine (m6A) modification of RNAs is the most pervasive and highly conserved RNA modification in eukaryotic cells; it exerts powerful impact on almost all aspects of mRNA processing that is mainly endowed by its binding with m6A reader proteins. In this study, we investigate the previously uncharacterized regulatory roles of YTHDC1, an m6A reader in mouse SCs. Our results demonstrate that YTHDC1 is an essential regulator of SC activation and proliferation upon acute injury-induced muscle regeneration. The induction of YTHDC1 is indispensable for SC activation and proliferation; thus, inducible YTHDC1 depletion almost abolishes SC regenerative capacity. Mechanistically, transcriptome-wide profiling using LACE-seq in both SCs and mouse C2C12 myoblasts identifies m6A-mediated binding targets of YTHDC1. Next, splicing analysis defines splicing mRNA targets of m6A-YTHDC1. Furthermore, nuclear export analysis also leads to the identification of potential mRNA export targets of m6A-YTHDC1 in SCs and C2C12 myoblasts;interestingly, some mRNAs can be regulated at both splicing and export levels. Lastly, we map YTHDC1 interacting protein partners in myoblasts and unveil a myriad of factors governing mRNA splicing, nuclear export, and transcription, among which hnRNPG appears to be a bona fide interacting partner of YTHDC1. Altogether, our findings uncover YTHDC1 as an essential factor controlling SC regenerative ability through multifaceted gene regulatory mechanisms in mouse myoblast cells.

Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation.

RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.

RBM46 is essential for gametogenesis and functions in post-transcriptional roles affecting meiotic cohesin subunits.

RBM46 is a germ cell-specific RNA-binding protein required for gametogenesis, but the targets and molecular functions of RBM46 remain unknown. Here, we demonstrate that RBM46 binds at specific motifs in the 3'UTRs of mRNAs encoding multiple meiotic cohesin subunits and show that RBM46 is required for normal synaptonemal complex formation during meiosis initiation. Using a recently reported, high-resolution technique known as LACE-seq and working with low-input cells, we profiled the targets of RBM46 at single-nucleotide resolution in leptotene and zygotene stage gametes. We found that RBM46 preferentially binds target mRNAs containing GCCUAU/GUUCGA motifs in their 3'UTRs regions. In Rbm46 knockout mice, the RBM46-target cohesin subunits displayed unaltered mRNA levels but had reduced translation, resulting in the failed assembly of axial elements, synapsis disruption, and meiotic arrest. Our study thus provides mechanistic insights into the molecular functions of RBM46 in gametogenesis and illustrates the power of LACE-seq for investigations of RNA-binding protein functions when working with low-abundance input materials.

Selective Translation of Maternal mRNA by eIF4E1B Controls Oocyte to Embryo Transition.

Maternal messenger ribonucleic acids (mRNAs) are driven by a highly orchestrated scheme of recruitment to polysomes and translational activation. However, selecting and regulating individual mRNAs for the translation from a competitive pool of mRNAs are little-known processes. This research shows that the maternal eukaryotic translation initiation factor 4e1b (Eif4e1b) expresses during the oocyte-to-embryo transition (OET), and maternal deletion of Eif4e1b leads to multiple defects concerning oogenesis and embryonic developmental competence during OET. The linear amplification of complementary deoxyribonucleic acid (cDNA) ends, and sequencing (LACE-seq) is used to identify the distinct subset of mRNA and its CG-rich binding sites within the 5' untranslated region (UTR) targeted by eIF4E1B. The proteomics analyses indicate that eIF4E1B-specific bound genes show stronger downregulation at the protein level, which further verify a group of proteins that plays a crucial role in oocyte maturation and embryonic developmental competence is insufficiently synthesized in Eif4e1b-cKO oocytes during OET. Moreover, the biochemical results in vitro are combined to further confirm the maternal-specific translation activation model assembled by eIF4E1B and 3'UTR-associated mRNA binding proteins. The findings demonstrate the indispensability of eIF4E1B for selective translation activation in mammalian oocytes and provide a potential network regulated by eIF4E1B in OET.