CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.

Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP, key steps in the PAR-CLIP procedure must be optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data analysis pipeline to three distinct RBPs targeting different annotation categories of cellular RNAs.

The placenta and decidua interact dynamically to enable embryonic and fetal development. Here, we report single-cell RNA sequencing of 14,341 and 6754 cells from first-trimester human placental villous and decidual tissues, respectively. Bioinformatic analysis identified major cell types, many known and some subtypes previously unknown in placental villi and decidual context. Further detailed analysis revealed proliferating subpopulations, enrichment of cell type-specific transcription factors, and putative intercellular communication in the fetomaternal microenvironment. This study provides a blueprint to further the understanding of the roles of these cells in the placenta and decidua for maintenance of early gestation as well as pathogenesis in pregnancy-related disorders.

Investigating human heart development and applying this to deviations resulting in disease is incomplete without molecular characterization of the cell types required for normal functioning. We investigated foetal human heart single-cell transcriptomes from mid-gestational healthy and anti-SSA/Ro associated congenital heart block (CHB) samples.

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis-acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors.IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter.

The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice. Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFβ superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.

Neuroendocrine neoplasms (NENs) are clinically diverse and incompletely characterized cancers that are challenging to classify. MicroRNAs (miRNAs) are small regulatory RNAs that can be used to classify cancers. Recently, a morphology-based classification framework for evaluating NENs from different anatomical sites was proposed by experts, with the requirement of improved molecular data integration. Here, we compiled 378 miRNA expression profiles to examine NEN classification through comprehensive miRNA profiling and data mining. Following data preprocessing, our final study cohort included 221 NEN and 114 non-NEN samples, representing 15 NEN pathological types and 5 site-matched non-NEN control groups. Unsupervised hierarchical clustering of miRNA expression profiles clearly separated NENs from non-NENs. Comparative analyses showed that miR-375 and miR-7 expression is substantially higher in NEN cases than non-NEN controls. Correlation analyses showed that NENs from diverse anatomical sites have convergent miRNA expression programs, likely reflecting morphological and functional similarities. Using machine learning approaches, we identified 17 miRNAs to discriminate 15 NEN pathological types and subsequently constructed a multilayer classifier, correctly identifying 217 (98%) of 221 samples and overturning one histological diagnosis. Through our research, we have identified common and type-specific miRNA tissue markers and constructed an accurate miRNA-based classifier, advancing our understanding of NEN diversity.

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell-specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell-specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.

The full set of microRNAs (miRNAs) in the human genome is not known. Because presently known miRNAs have been identified by virtue of their abundant expression in a few cell types, many tissue-specific miRNAs remain unrevealed. To understand the role of miRNAs in B cell function and lymphomagenesis, we generated short-RNA libraries from normal human B cells at different stages of development (naive, germinal center, memory) and from a Burkitt lymphoma cell line. A combination of cloning and computational analysis identified 178 miRNAs (miRNome) expressed in normal and/or transformed B cell libraries. Most notably, the B cell miRNome included 75 miRNAs which to our knowledge have not been previously reported and of which 66 have been validated by RNA blot and/or RT-PCR analyses. Numerous miRNAs were expressed in a stage- or transformation-specific fashion in B cells, suggesting specific functional or pathologic roles. These results provide a resource for studying the role of miRNAs in B cell development, immune function, and lymphomagenesis.

Autosomal dominant polycystic kidney disease (ADPKD) is clinically heterogenic. Biomarkers are needed to predict prognosis and guide management. We aimed to profile microRNA (miRNA) in ADPKD to gain molecular insight and evaluate biomarker potential.

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

Atopic dermatitis (AD) is a prevalent inflammatory skin disease with a complex pathogenesis involving immune cell and epidermal abnormalities. Despite whole tissue biopsy studies that have advanced the mechanistic understanding of AD, single cell-based molecular alterations are largely unknown.

The characterization of post-transcriptional gene regulation by small regulatory RNAs of 20-30 nt length, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for the characterization of small RNAs is their identification and quantification across different developmental stages, normal and diseased tissues, as well as model cell lines. Here we present a step-by-step protocol for the bioinformatic analysis of barcoded cDNA libraries for small RNA profiling generated by Illumina sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections.

On a retrospective cohort of 1,082 FFPE breast tumors, we demonstrated the analytical validity of a test using multiplexed RNA-FISH-guided laser capture microdissection (LCM) coupled with RNA-sequencing (mFISHseq), which showed 93% accuracy compared to immunohistochemistry. The combination of these technologies makes strides in i) precisely assessing tumor heterogeneity, ii) obtaining pure tumor samples using LCM to ensure accurate biomarker expression and multigene testing, and iii) providing thorough and granular data from whole transcriptome profiling. We also constructed a 293-gene intrinsic subtype classifier that performed equivalent to the research based PAM50 and AIMS classifiers. By combining three molecular classifiers for consensus subtyping, mFISHseq alleviated single sample discordance, provided near perfect concordance with other classifiers (κ > 0.85), and reclassified 30% of samples into different subtypes with prognostic implications. We also use a consensus approach to combine information from 4 multigene prognostic classifiers and clinical risk to characterize high, low, and ultra-low risk patients that relapse early (< 5 years), late (> 10 years), and rarely, respectively. Lastly, to identify potential patient subpopulations that may be responsive to treatments like antibody drug-conjugates (ADC), we curated a list of 92 genes and 110 gene signatures to interrogate their association with molecular subtype and overall survival. Many genes and gene signatures related to ADC processing (e.g., antigen/payload targets, endocytosis, and lysosome activity) were independent predictors of overall survival in multivariate Cox regression models, thus highlighting potential ADC treatment-responsive subgroups. To test this hypothesis, we constructed a unique 19-feature classifier using multivariate logistic regression with elastic net that predicted response to trastuzumab emtansine (T-DM1; AUC = 0.96) better than either ERBB2 mRNA or Her2 IHC alone in the T-DM1 arm of the I-SPY2 trial. This test was deployed in a research-use only format on 26 patients and revealed clinical insights into patient selection for novel therapies like ADCs and immunotherapies and de-escalation of adjuvant chemotherapy.

Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.