Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Capturing the cloud: UAP56 in nuage assembly and function.

The nuage is a hazy electron-dense structure unique to germ cells and is enriched in components of the piRNA pathway. Although the nuage is cytoplasmic, Zhang et al. now show that it is organized by an intranuclear protein, UAP56.

Post-transcriptional regulation of mouse neurogenesis by Pumilio proteins.

Despite extensive studies on mammalian neurogenesis, its post-transcriptional regulation remains under-explored. Here we report that neural-specific inactivation of two murine post-transcriptional regulators, Pumilio 1 (Pum1) and Pum2, severely reduced the number of neural stem cells (NSCs) in the postnatal dentate gyrus (DG), drastically increased perinatal apoptosis, altered DG cell composition, and impaired learning and memory. Consistently, the mutant DG neurospheres generated fewer NSCs with defects in proliferation, survival, and differentiation, supporting a major role of Pum1 and Pum2 in hippocampal neurogenesis and function. Cross-linking immunoprecipitation revealed that Pum1 and Pum2 bind to thousands of mRNAs, with at least 694 common targets in multiple neurogenic pathways. Depleting Pum1 and/or Pum2 did not change the abundance of most target mRNAs but up-regulated their proteins, indicating that Pum1 and Pum2 regulate the translation of their target mRNAs. Moreover, Pum1 and Pum2 display RNA-dependent interaction with fragile X mental retardation protein (FMRP) and bind to one another's mRNA. This indicates that Pum proteins might form collaborative networks with FMRP and possibly other post-transcriptional regulators to regulate neurogenesis.

MIWI prevents aneuploidy during meiosis by cleaving excess satellite RNA.

MIWI, a murine member of PIWI proteins mostly expressed during male meiosis, is crucial for piRNA biogenesis, post-transcriptional regulation, and spermiogenesis. However, its meiotic function remains unknown. Here, we report that MIWI deficiency alters meiotic kinetochore assembly, significantly increases chromosome misalignment at the meiosis metaphase I plate, and causes chromosome mis-segregation. Consequently, Miwi-deficient mice show elevated aneuploidy in metaphase II and spermatid death. Furthermore, in Miwi-null and Miwi slicer-deficient mutants, major and minor satellite RNAs from centromeric and pericentromeric satellite repeats accumulate in excess. Over-expression of satellite repeats in wild-type spermatocytes also causes elevated chromosome misalignment, whereas reduction of both strands of major or minor satellite RNAs results in lower frequencies of chromosome misalignment. We show that MIWI, guided by piRNA, cleaves major satellite RNAs, generating RNA fragments that may form substrates for subsequent Dicer cleavage. Furthermore, Dicer cleaves all satellite RNAs in conjunction with MIWI. These findings reveal a novel mechanism in which MIWI- and Dicer-mediated cleavage of the satellite RNAs prevents the over-expression of satellite RNAs, thus ensuring proper kinetochore assembly and faithful chromosome segregation during meiosis.

Artificial intelligence-assisted system for the assessment of Forrest classification of peptic ulcer bleeding: a multicenter diagnostic study.

BACKGROUND: Inaccurate Forrest classification may significantly affect clinical outcomes, especially in high risk patients. Therefore, this study aimed to develop a real-time deep convolutional neural network (DCNN) system to assess the Forrest classification of peptic ulcer bleeding (PUB). METHODS: A training dataset (3868 endoscopic images) and an internal validation dataset (834 images) were retrospectively collected from the 900th Hospital, Fuzhou, China. In addition, 521 images collected from four other hospitals were used for external validation. Finally, 46 endoscopic videos were prospectively collected to assess the real-time diagnostic performance of the DCNN system, whose diagnostic performance was also prospectively compared with that of three senior and three junior endoscopists. RESULTS: The DCNN system had a satisfactory diagnostic performance in the assessment of Forrest classification, with an accuracy of 91.2% (95%CI 89.5%-92.6%) and a macro-average area under the receiver operating characteristic curve of 0.80 in the validation dataset. Moreover, the DCNN system could judge suspicious regions automatically using Forrest classification in real-time videos, with an accuracy of 92.0% (95%CI 80.8%-97.8%). The DCNN system showed more accurate and stable diagnostic performance than endoscopists in the prospective clinical comparison test. This system helped to slightly improve the diagnostic performance of senior endoscopists and considerably enhance that of junior endoscopists. CONCLUSION: The DCNN system for the assessment of the Forrest classification of PUB showed satisfactory diagnostic performance, which was slightly superior to that of senior endoscopists. It could therefore effectively assist junior endoscopists in making such diagnoses during gastroscopy.

The biogenesis and function of PIWI proteins and piRNAs: progress and prospect.

The evolutionarily conserved Argonaute/PIWI (AGO/PIWI, also known as PAZ-PIWI domain or PPD) family of proteins is crucial for the biogenesis and function of small noncoding RNAs (ncRNAs). This family can be divided into AGO and PIWI subfamilies. The AGO proteins are ubiquitously present in diverse tissues. They bind to small interfering RNAs (siRNAs) and microRNAs (miRNAs). In contrast, the PIWI proteins are predominantly present in the germline and associate with a novel class of small RNAs known as PIWI-interacting RNAs (piRNAs). Tens of thousands of piRNA species, typically 24-32 nucleotide (nt) long, have been found in mammals, zebrafish, and Drosophila. Most piRNAs appear to be generated from a small number of long single-stranded RNA precursors that are often encoded by repetitive intergenic sequences in the genome. PIWI proteins play crucial roles during germline development and gametogenesis of many metazoan species, from germline determination and germline stem cell (GSC) maintenance to meiosis, spermiogenesis, and transposon silencing. These diverse functions may involve piRNAs and may be achieved via novel mechanisms of epigenetic and posttranscriptional regulation.

PIWIL1 promotes gastric cancer via a piRNA-independent mechanism.

Targeted cancer therapy aims to achieve specific elimination of cancerous but not normal cells. Recently, PIWI proteins, a subfamily of the PAZ-PIWI domain (PPD) protein family, have emerged as promising candidates for targeted cancer therapy. PPD proteins are essential for small noncoding RNA pathways. The Argonaute subfamily partners with microRNA and small interfering RNA, whereas the PIWI subfamily partners with PIWI-interacting RNA (piRNA). Both PIWI proteins and piRNA are mostly expressed in the germline and best known for their function in transposon silencing, with no detectable function in mammalian somatic tissues. However, PIWI proteins become aberrantly expressed in multiple types of somatic cancers, thus gaining interest in targeted therapy. Despite this, little is known about the regulatory mechanism of PIWI proteins in cancer. Here we report that one of the four PIWI proteins in humans, PIWIL1, is highly expressed in gastric cancer tissues and cell lines. Knocking out the PIWIL1 gene (PIWIL1-KO) drastically reduces gastric cancer cell proliferation, migration, metastasis, and tumorigenesis. RNA deep sequencing of gastric cancer cell line SNU-1 reveals that KO significantly changes the transcriptome, causing the up-regulation of most of its associated transcripts. Surprisingly, few bona fide piRNAs exist in gastric cancer cells. Furthermore, abolishing the piRNA-binding activity of PIWIL1 does not affect its oncogenic function. Thus, PIWIL1 function in gastric cancer cells is independent of piRNA. This piRNA-independent regulation involves interaction with the UPF1-mediated nonsense-mediated mRNA decay (NMD) mechanism. Altogether, our findings reveal a piRNA-independent function of PIWIL1 in promoting gastric cancer.

Pumilio proteins utilize distinct regulatory mechanisms to achieve complementary functions required for pluripotency and embryogenesis.

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.

Deciphering the role of RNA structure in translation efficiency.

BACKGROUND: RNA secondary structure has broad impact on the fate of RNA metabolism. The reduced stability of secondary structures near the translation initiation site/start codon of the coding region promotes the efficiency of translation in both prokaryotic and eukaryotic species. However, the inaccuracy of in silico folding and the focus on the coding region limit our understanding of the global relationship between the whole mRNA structure and translation efficiency. Leveraging high-throughput RNA structure probing data in the transcriptome, we aim to systematically investigate the role of RNA structure in regulating translation efficiency. RESULTS: Here, we analyze the influences of hundreds of sequence and structural features on translation efficiency in the mouse embryonic stem cells (mESCs) and zebrafish developmental stages. Our findings reveal that overall in vivo RNA structure has a higher relative importance in predicting translation efficiency than in vitro RNA structure in both mESCs and zebrafish. Also, RNA structures in 3' untranslated region (UTR) have much stronger influence on translation efficiency compared to those in coding regions or 5' UTR. Furthermore, strong alternation between in vitro and in vivo structures in 3' UTR are detected in highly translated mRNAs in mESCs but not zebrafish. Instead, moderate alteration between in vitro and in vivo RNA structures in the 5' UTR and proximal coding regions are detected in highly translated mRNAs in zebrafish. CONCLUSIONS: Our results suggest the openness of the 3' UTR promotes the translation efficiency in both mice and zebrafish, with the in vivo structure in 3' UTR more important in mice than in zebrafish. This reveals a novel role of RNA secondary structure on translational regulation.

MIWI2 targets RNAs transcribed from piRNA-dependent regions to drive DNA methylation in mouse prospermatogonia.

Argonaute/Piwi proteins can regulate gene expression via RNA degradation and translational regulation using small RNAs as guides. They also promote the establishment of suppressive epigenetic marks on repeat sequences in diverse organisms. In mice, the nuclear Piwi protein MIWI2 and Piwi-interacting RNAs (piRNAs) are required for DNA methylation of retrotransposon sequences and some other sequences. However, its underlying molecular mechanisms remain unclear. Here, we show that piRNA-dependent regions are transcribed at the stage when piRNA-mediated DNA methylation takes place. MIWI2 specifically interacts with RNAs from these regions. In addition, we generated mice with deletion of a retrotransposon sequence either in a representative piRNA-dependent region or in a piRNA cluster. Both deleted regions were required for the establishment of DNA methylation of the piRNA-dependent region, indicating that piRNAs determine the target specificity of MIWI2-mediated DNA methylation. Our results indicate that MIWI2 affects the chromatin state through base-pairing between piRNAs and nascent RNAs, as observed in other organisms possessing small RNA-mediated epigenetic regulation.

PIWI-piRNA pathway-mediated transposable element repression in Hydra somatic stem cells.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI-piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI-piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI-piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI-piRNA pathway.

Posttranscriptional regulation of gene expression by Piwi proteins and piRNAs.

Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for gametogenesis, embryogenesis, and stem cell maintenance in animals. Piwi proteins act on transposon RNAs by cleaving the RNAs and by interacting with factors involved in RNA regulation. Additionally, piRNAs generated from transposons and psuedogenes can be used by Piwi proteins to regulate mRNAs at the posttranscriptional level. Here we discuss piRNA biogenesis, recent findings on posttranscriptional regulation of mRNAs by the piRNA pathway, and the potential importance of this posttranscriptional regulation for a variety of biological processes such as gametogenesis, developmental transitions, and sex determination.

MicroRNAs: key regulators of stem cells.

The hallmark of a stem cell is its ability to self-renew and to produce numerous differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new dimension of gene regulation in controlling stem cell fate and behaviour.

Heat shock protein DNAJA1 stabilizes PIWI proteins to support regeneration and homeostasis of planarian Schmidtea mediterranea.

PIWI proteins are key regulators of germline and somatic stem cells throughout different evolutionary lineages. However, how PIWI proteins themselves are regulated remains largely unknown. To identify candidate proteins that interact with PIWI proteins and regulate their stability, here we established a yeast two-hybrid (Y2H) assay in the planarian species Schmidtea mediterranea We show that DNAJA1, a heat shock protein 40 family member, interacts with the PIWI protein SMEDWI-2, as validated by the Y2H screen and co-immunoprecipitation assays. We found that DNAJA1 is enriched in planarian adult stem cells, the nervous system, and intestinal tissues. DNAJA1-knockdown abolished planarian regeneration and homeostasis, compromised stem cell maintenance and PIWI-interacting RNA (piRNA) biogenesis, and deregulated SMEDWI-1/2 target genes. Mechanistically, we observed that DNAJA1 is required for the stability of SMEDWI-1 and SMEDWI-2 proteins. Furthermore, we noted that human DNAJA1 binds to Piwi-like RNA-mediated gene silencing 1 (PIWIL1) and is required for PIWIL1 stability in human gastric cancer cells. In summary, our results reveal not only an evolutionarily conserved functional link between PIWI and DNAJA1 that is essential for PIWI protein stability and piRNA biogenesis, but also an important role of DNAJA1 in the control of proteins involved in stem cell regulation.

PIWI proteins and PIWI-interacting RNAs in the soma.

The discovery of millions of PIWI-interacting RNAs revealed a fascinating and unanticipated dimension of biology. The PIWI-piRNA pathway has been commonly perceived as germline-specific, even though the somatic function of PIWI proteins was documented when they were first discovered. Recent studies have begun to re-explore this pathway in somatic cells in diverse organisms, particularly lower eukaryotes. These studies have illustrated the multifaceted somatic functions of the pathway not only in transposon silencing but also in genome rearrangement and epigenetic programming, with biological roles in stem-cell function, whole-body regeneration, memory and possibly cancer.

A critical role for nucleoporin 358 (Nup358) in transposon silencing and piRNA biogenesis in Drosophila.

Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)-dependent ping-pong cycle generates most germline piRNAs. Loading of antisense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.

Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms.

The topipotency of the germline is the full manifestation of the pluri- and multipotency of embryonic and adult stem cells, thus the germline and stem cells must share common mechanisms that guarantee their multipotentials in development. One of the few such known shared mechanisms is represented by Piwi proteins, which constitute one of the two subfamilies of the Argonaute protein family. Piwi proteins bind to Piwi-interacting RNAs (piRNAs) that are generally 26 to 31 nucleotides in length. Both Piwi proteins and piRNAs are most abundantly expressed in the germline. Moreover, Piwi proteins are expressed broadly in certain types of somatic stem/progenitor cells and other somatic cells across animal phylogeny. Recent studies indicate that the Piwi-piRNA pathway mediates epigenetic programming and posttranscriptional regulation, which may be responsible for its function in germline specification, gametogenesis, stem cell maintenance, transposon silencing, and genome integrity in diverse organisms.

Tudor-SN Interacts with Piwi Antagonistically in Regulating Spermatogenesis but Synergistically in Silencing Transposons in Drosophila.

Piwi proteins associate with piRNAs and functions in epigenetic programming, post-transcriptional regulation, transposon silencing, and germline development. However, it is not known whether the diverse functions of these proteins are molecularly separable. Here we report that Piwi interacts with Tudor-SN (Tudor staphylococcal nuclease, TSN) antagonistically in regulating spermatogenesis but synergistically in silencing transposons. However, it is not required for piRNA biogenesis. TSN is known to participate in diverse molecular functions such as RNAi, degradation of hyper-edited miRNAs, and spliceosome assembly. We show that TSN colocalizes with Piwi in primordial germ cells (PGCs) and embryonic somatic cells. In adult ovaries and testes, TSN is ubiquitously expressed and enriched in the cytoplasm of both germline and somatic cells. The tsn mutants display a higher mitotic index of spermatogonia, accumulation of spermatocytes, defects in meiotic cytokinesis, a decreased number of spermatids, and eventually reduced male fertility. Germline-specific TSN-expression analysis demonstrates that this function is germline-dependent. Different from other known Piwi interters, TSN represses Piwi expression at both protein and mRNA levels. Furthermore, reducing piwi expression in the germline rescues tsn mutant phenotype in a dosage-dependent manner, demonstrating that Piwi and TSN interact antagonistically in germ cells to regulate spermatogenesis. However, the tsn deficiency has little, if any, impact on piRNA biogenesis but displays a synergistic effect with piwi mutants in transposon de-silencing. Our results reveal the biological function of TSN and its contrasting modes of interaction with Piwi in spermatogenesis, transposon silencing, and piRNA biogenesis.

Noncoding RNAs in the regulation of DNA replication.

Noncoding RNAs (ncRNAs) have crucial roles in epigenetic, transcriptional, and post-transcriptional regulation. Recent studies have begun to reveal a role of ncRNAs in DNA replication. Here, we review the roles of ncRNAs in regulating different aspects of DNA replication in prokaryotic and eukaryotic systems. We speculate that ncRNAs might function to guide the origin recognition complex (ORC) to chromosomal DNA during replication initiation in higher eukaryotes.