Mammalian 5'-capped microRNA precursors that generate a single microRNA.

MicroRNAs (miRNAs) are short RNA gene regulators typically produced from primary transcripts that are cleaved by the nuclear microprocessor complex, with the resulting precursor miRNA hairpins exported by exportin 5 and processed by cytoplasmic Dicer to yield two (5p and 3p) miRNAs. Here, we document microprocessor-independent 7-methylguanosine (m(7)G)-capped pre-miRNAs, whose 5' ends coincide with transcription start sites and 3' ends are most likely generated by transcription termination. By establishing a small RNA Cap-seq method that employs the cap-binding protein eIF4E, we identified a group of murine m(7)G-capped pre-miRNAs genome wide. The m(7)G-capped pre-miRNAs are exported via the PHAX-exportin 1 pathway. After Dicer cleavage, only the 3p-miRNA is efficiently loaded onto Argonaute to form a functional microRNP. This unusual miRNA biogenesis pathway, which differs in pre-miRNA synthesis, nuclear-cytoplasmic transport, and guide strand selection, enables the development of shRNA expression constructs that produce a single 3p-siRNA.

Widespread Inducible Transcription Downstream of Human Genes.

Pervasive transcription of the human genome generates RNAs whose mode of formation and functions are largely uncharacterized. Here, we combine RNA-seq with detailed mechanistic studies to describe a transcript type derived from protein-coding genes. The resulting RNAs, which we call DoGs for downstream of gene containing transcripts, possess long non-coding regions (often >45 kb) and remain chromatin bound. DoGs are inducible by osmotic stress through an IP3 receptor signaling-dependent pathway, indicating active regulation. DoG levels are increased by decreased termination of the upstream transcript, a previously undescribed mechanism for rapid transcript induction. Relative depletion of polyA signals in DoG regions correlates with increased levels of DoGs after osmotic stress. We detect DoG transcription in several human cell lines and provide evidence for thousands of DoGs genome wide.

Integrated genomic characterization of ERBB2/HER2 alterations in invasive breast carcinoma: a focus on unusual FISH groups.

In patients with invasive breast cancer, fluorescence in situ hybridization (FISH) testing for HER2 typically demonstrates the clear presence or lack of ERBB2 (HER2) amplification (i.e., groups 1 or 5). However, a small subset of patients can present with unusual HER2 FISH patterns (groups 2-4), resulting in diagnostic confusion. To provide clarity, the 2018 CAP/ASCO HER2 testing guideline recommends additional testing using HER2 immunohistochemistry (IHC) for determining the final HER2 status. Despite this effort, the genomic correlates of unusual HER2 FISH groups remain poorly understood. Here, we used droplet digital PCR (ddPCR) and targeted next-generation sequencing (NGS) to characterize the genomic features of both usual and unusual HER2 FISH groups. In this study, 51 clinical samples were selected to represent FISH groups 1-5. Furthermore, group 1 was subdivided into two groups, with groups 1A and 1B corresponding to cases with HER2 signals/cell ≥6.0 and 4-6, respectively. Overall, our findings revealed a wide range of copy number alterations in HER2 across the different FISH groups. As expected, groups 1A and 5 showed the clear presence and lack of HER2 copy number gain, respectively, as measured by ddPCR and NGS. In contrast, group 1B and other uncommon FISH groups (groups 2-4) were characterized by a broader range of HER2 copy levels with only a few select cases showing high-level gain. Notably, these cases with increased HER2 copy levels also showed HER2 overexpression by IHC, thus highlighting the correlation between HER2 copy number and HER2 protein expression. Given the concordance between the genomic and protein results, our findings suggest that HER2 IHC may inform HER2 copy number status in patients with unusual FISH patterns. Hence, our results support the current recommendation for using IHC to resolve HER2 status in FISH groups 2-4.

Comparative analysis reveals genomic features of stress-induced transcriptional readthrough.

Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in stress responses and adaptation. Genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of RNA classes, most of unknown function. One such class, termed downstream of gene-containing transcripts (DoGs), was reported to result from transcriptional readthrough upon osmotic stress in human cells. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a genome-wide mapping of transcriptional readthrough, using nuclear RNA-Seq, comparing heat shock, osmotic stress, and oxidative stress in NIH 3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough, both in levels and length, under all stress conditions, with significant, yet not complete, overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Analysis of public datasets detected increases in polymerase II occupancy in DoG regions after heat shock, supporting our findings. Interestingly, DoGs tend to be produced in the vicinity of neighboring genes, leading to a marked increase in their antisense-generating potential. Finally, we examine genomic features of readthrough transcription and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state.

Caution needs to be taken when assigning transcription start sites to ends of protein-coding genes: a rebuttal.

Naturally occurring stress-induced transcriptional readthrough is a recently discovered phenomenon, in which stress conditions lead to dramatic induction of long transcripts as a result of transcription termination failure. In 2015, we reported the induction of such downstream of gene (DoG) containing transcripts upon osmotic stress in human cells, while others observed similar transcripts in virus-infected and cancer cells. Using the rigorous methodology Cap-Seq, we demonstrated that DoGs result from transcriptional readthrough, not de novo initiation. More recently, we presented a genome-wide comparison of NIH3T3 mouse cells subjected to osmotic, heat, and oxidative stress and concluded that massive induction of transcriptional readthrough is a hallmark of the mammalian stress response. In their recent letter, Huang and Liu in contrast claim that DoG transcripts result from novel transcription initiation near the ends of genes. Their conclusions rest on analyses of a publicly available transcription start site (TSS-Seq) dataset from unstressed NIH3T3 cells. Here, we present evidence that this dataset identifies not only true transcription start sites, TSSs, but also 5'-ends of numerous snoRNAs, which are generally processed from introns in mammalian cells. We show that failure to recognize these erroneous assignments in the TSS-Seq dataset, as well as ignoring published Cap-Seq data on TSS mapping during osmotic stress, have led to misinterpretation by Huang and Liu. We conclude that, contrary to the claims made by Huang and Liu, TSS-Seq reads near gene ends cannot explain the existence of DoGs, nor their stress-mediated induction. Rather it is, as we originally demonstrated, transcriptional readthrough that leads to the formation of DoGs.

Readthrough transcription: How are DoGs made and what do they do?

In recent years, the realization that most of the genome is transcribed has transformed the study of mammalian gene expression. Much effort has gone into investigating how this pervasive transcription is regulated and what the functions of the resulting transcripts are, if any. We recently discovered that stress-induced transcriptional readthrough generates very long downstream of gene containing transcripts (DoGs), which may explain up to 20% of intergenic transcription. DoGs are induced by osmotic stress at the level of transcription by a mechanism that depends on calcium release from the endoplasmic reticulum mediated by IP3 receptors. Here, we discuss DoG induction and function in the context of the literature, with special focus on 2 outstanding questions. First, we discuss possible molecular mechanisms underlying DoG induction through reduced transcription termination. Second, we explore how DoGs may function in maintaining euchromatin after nuclear scaffold stress. In short, we review important aspects of DoG biogenesis and function, and provide an outlook for continued DoG study.

The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element.

The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3' UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA.

The p53 target protein Wig-1 binds hnRNP A2/B1 and RNA Helicase A via RNA.

The p53-induced Wig-1 gene encodes a double stranded RNA-binding zinc finger protein. We generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. Induction of Wig-1 expression by doxycycline inhibited cell growth in a long-term assay but did not cause any changes in cell cycle distribution nor increased fraction of apoptotic cells. Using co-immunoprecipitation and mass spectrometry, we identified two Wig-1-binding proteins, hnRNP A2/B1 and RNA Helicase A, both of which are involved in RNA processing. The binding was dependent on the presence of RNA. Our results establish a link between the p53 tumor suppressor and RNA processing via hnRNPA2/B1 and RNA Helicase A.

A robust targeted sequencing approach for low input and variable quality DNA from clinical samples.

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification-this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.

Calcium signaling and transcription: elongation, DoGs, and eRNAs.

The calcium ion (Ca2+) is a key intracellular signaling molecule with far-reaching effects on many cellular processes. One of the most important such Ca2+ regulated processes is transcription. A body of literature describes the effect of Ca2+ signaling on transcription initiation as occurring mainly through activation of gene-specific transcription factors by Ca2+-induced signaling cascades. However, the reach of Ca2+ extends far beyond the first step of transcription. In fact, Ca2+ can regulate all phases of transcription, with additional effects on transcription-associated events such as alternative splicing. Importantly, Ca2+ signaling mediates reduced transcription termination in response to certain stress conditions. This reduction allows readthrough transcription, generating a highly inducible and diverse class of downstream of gene containing transcripts (DoGs) that we have recently described.

Genome-wide identification of Wig-1 mRNA targets by RIP-Seq analysis.

RNA-binding proteins (RBPs) play important roles in the regulation of gene expression through a variety of post-transcriptional mechanisms. The p53-induced RBP Wig-1 (Zmat3) binds RNA through its zinc finger domains and enhances stability of p53 and N-Myc mRNAs and decreases stability of FAS mRNA. To identify novel Wig-1-bound RNAs, we performed RNA-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) in HCT116 and Saos-2 cells. We identified 286 Wig-1-bound mRNAs common between the two cell lines. Sequence analysis revealed that AU-rich elements (AREs) are highly enriched in the 3'UTR of these Wig-1-bound mRNAs. Network enrichment analysis showed that Wig-1 preferentially binds mRNAs involved in cell cycle regulation. Moreover, we identified a 2D Wig-1 binding motif in HIF1A mRNA. Our findings confirm that Wig-1 is an ARE-BP that regulates cell cycle-related processes and provide a novel view of how Wig-1 may bind mRNA through a putative structural motif. We also significantly extend the repertoire of Wig-1 target mRNAs. Since Wig-1 is a transcriptional target of the tumor suppressor p53, these results have implications for our understanding of p53-dependent stress responses and tumor suppression.

Regulation of tumor suppressor p53 at the RNA level.

p53 is a key tumor suppressor that triggers cell cycle arrest, senescence, or apoptosis in response to cellular stress. Frequent p53 mutation in human tumors allows survival, sustained growth, and tumor progression. p53 is expressed at low levels under normal conditions, due to rapid protein turnover. Stress signaling induces p53 protein stabilization through phosphorylation and other post-translational modifications. However, recent studies have demonstrated critical regulation of p53 at the mRNA level, mediated via both the 5'UTR and the 3'UTR and affecting both the stability and the translation efficiency of the p53 mRNA. Both proteins and microRNAs have been implicated in such regulation. The p53 target gene Wig-1 encodes a zinc finger protein that binds to double-stranded RNA and enhances p53 mRNA stability by binding to the 3'UTR in a positive feedback loop. Here, we shall summarize current knowledge about regulation of the p53 mRNA and discuss possible implications for cancer therapy.