Sarah F. Newbury

Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma

Background:Multiple myeloma is a plasma cell disorder that is characterised by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in the blood or urine and associated organ dysfunction. It accounts for approximately 1% of cancers and 13% of haematological cancers. Myeloma arises from an asymptomatic proliferation of monoclonal plasma cells termed monoclonal gammopathy of undetermined significance (MGUS).Methods:MicroRNA expression profiling of serum samples was performed on three patient groups as well as normal controls. Validation of the nine microRNAs detected as promising biomarkers was carried out using TaqMan quantitative reverse transcription PCR. MicroRNA levels in serum were normalised using standard curves to determine the numbers of microRNAs per μl of serum.Results:Three serum microRNAs, miR-720, miR-1308 and miR-1246, were found to have potential as diagnostic biomarkers in myeloma. Use of miR-720 and miR-1308 together provides a powerful diagnostic tool for distinguishing normal healthy controls, as well as patients with unrelated illnesses, from pre-cancerous myeloma and myeloma patients. In addition, the combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients.Conclusion:We have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for multiple myeloma.

The 5' → 3' exoribonuclease XRN1/Pacman and its functions in cellular processes and development.

XRN1 is a 5′ → 3′ processive exoribonuclease that degrades mRNAs after they have been decapped. It is highly conserved in all eukaryotes, including homologs in Drosophila melanogaster (Pacman), Caenorhabditis elegans (XRN1), and Saccharomyces cerevisiae (Xrn1p). As well as being a key enzyme in RNA turnover, XRN1 is involved in nonsense‐mediated mRNA decay and degradation of mRNAs after they have been targeted by small interfering RNAs or microRNAs. The crystal structure of XRN1 can explain its processivity and also the selectivity of the enzyme for 5′ monophosphorylated RNA. In eukaryotic cells, XRN1 is often found in particles known as processing bodies (P bodies) together with other proteins involved in the 5′ → 3′ degradation pathway, such as DCP2 and the helicase DHH1 (Me31B). Although XRN1 shows little specificity to particular 5′ monophosphorylated RNAs in vitro, mutations in XRN1 in vivo have specific phenotypes suggesting that it specifically degrades a subset of RNAs. In Drosophila, mutations in the gene encoding the XRN1 homolog pacman result in defects in wound healing, epithelial closure and stem cell renewal in testes. We propose a model where specific mRNAs are targeted to XRN1 via specific binding of miRNAs and/or RNA‐binding proteins to instability elements within the RNA. These guide the RNA to the 5′ core degradation apparatus for controlled degradation. WIREs RNA 2012, 3:455–468. doi: 10.1002/wrna.1109

Circulating Plasma microRNAs can differentiate Human Sepsis and Systemic Inflammatory Response Syndrome (SIRS).

Systemic inflammation in humans may be triggered by infection, termed sepsis, or non-infective processes, termed non-infective systemic inflammatory response syndrome (SIRS). MicroRNAs regulate cellular processes including inflammation and may be detected in blood. We aimed to establish definitive proof-of-principle that circulating microRNAs are differentially affected during sepsis and non-infective SIRS. Critically ill patients with severe (n = 21) or non-severe (n = 8) intra-abdominal sepsis; severe (n = 23) or non-severe (n = 21) non-infective SIRS; or no SIRS (n = 16) were studied. Next-generation sequencing and qRT-PCR were used to measure plasma microRNAs. Detectable blood miRNAs (n = 116) were generally up-regulated in SIRS compared to no-SIRS patients. Levels of these ‘circulating inflammation-related microRNAs’ (CIR-miRNAs) were 2.64 (IQR: 2.10–3.29) and 1.52 (IQR: 1.15–1.92) fold higher for non-infective SIRS and sepsis respectively (p < 0.0001), hence CIR-miRNAs appeared less abundant in sepsis than in SIRS. Six CIR-miRNAs (miR-30d-5p, miR-30a-5p, miR-192-5p, miR-26a-5p, miR-23a-5p, miR-191-5p) provided good-to-excellent discrimination of severe sepsis from severe SIRS (0.742–0.917 AUC of ROC curves). CIR-miRNA levels inversely correlated with pro-inflammatory cytokines (IL-1, IL-6 and others). Thus, among critically ill patients, sepsis and non-infective SIRS are associated with substantial, differential changes in CIR-miRNAs. CIR-miRNAs may be regulators of inflammation and warrant thorough evaluation as diagnostic and therapeutic targets.

The 5'-3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered

Background information. Ribonucleases have been well studied in yeast and bacteria, but their biological significance to developmental processes in multicellular organisms is not well understood. However, there is increasing evidence that specific timed transcript degradation is critical for regulation of many cellular processes, including translational repression, nonsense‐mediated decay and RNA interference. The Drosophila gene pacman is highly homologous to the major yeast exoribonuclease XRN1 and is the only known cytoplasmic 5′–3′ exoribonuclease in eukaryotes. To determine the effects of this exoribonuclease in development we have constructed a number of mutations in pacman by P‐element excision and characterized the resulting phenotypes.

Xrn1/Pacman affects apoptosis and regulates expression of hid and reaper.

Programmed cell death, or apoptosis, is a highly conserved cellular process that is crucial for tissue homeostasis under normal development as well as environmental stress. Misregulation of apoptosis is linked to many developmental defects and diseases such as tumour formation, autoimmune diseases and neurological disorders. In this paper, we show a novel role for the exoribonuclease Pacman/Xrn1 in regulating apoptosis. Using Drosophila wing imaginal discs as a model system, we demonstrate that a null mutation in pacman results in small imaginal discs as well as lethality during pupation. Mutant wing discs show an increase in the number of cells undergoing apoptosis, especially in the wing pouch area. Compensatory proliferation also occurs in these mutant discs, but this is insufficient to compensate for the concurrent increase in apoptosis. The phenotypic effects of the pacman null mutation are rescued by a deletion that removes one copy of each of the pro-apoptotic genes reaper, hid and grim, demonstrating that pacman acts through this pathway. The null pacman mutation also results in a significant increase in the expression of the pro-apoptotic mRNAs, hid and reaper, with this increase mostly occurring at the post-transcriptional level, suggesting that Pacman normally targets these mRNAs for degradation. Our results uncover a novel function for the conserved exoribonuclease Pacman and suggest that this exoribonuclease is important in the regulation of apoptosis in other organisms.

The 5'-3' exoribonuclease Pacman (Xrn1) regulates expression of the heat shock protein Hsp67Bc and the microRNA miR-277-3p in Drosophila wing imaginal discs

Pacman/Xrn1 is a highly conserved exoribonuclease known to play a critical role in gene regulatory events such as control of mRNA stability, RNA interference and regulation via miRNAs. Although Pacman has been well studied in Drosophila tissue culture cells, the biologically relevant cellular pathways controlled by Pacman in natural tissues are unknown. This study shows that a hypomorphic mutation in pacman (pcm5) results in smaller wing imaginal discs. These tissues, found in the larva, are known to grow and differentiate to form wing and thorax structures in the adult fly. Using microarray analysis, followed by quantitative RT-PCR, we show that eight mRNAs were increased in level by > 2-fold in the pcm5 mutant wing discs compared with the control. The levels of pre-mRNAs were tested for five of these mRNAs; four did not increase in the pcm5 mutant, showing that they are regulated at the post-transcriptional level and, therefore, could be directly affected by Pacman. These transcripts include one that encodes the heat shock protein Hsp67Bc, which is upregulated 11.9-fold at the post-transcriptional level and 2.3-fold at the protein level. One miRNA, miR-277-3p, is 5.6-fold downregulated at the post-transcriptional level in mutant discs, suggesting that Pacman affects its processing in this tissue. Together, these data show that a relatively small number of mRNAs and miRNAs substantially change in abundance in pacman mutant wing imaginal discs. Since Hsp67Bc is known to regulate autophagy and protein synthesis, it is possible that Pacman may control the growth of wing imaginal discs by regulating these processes.

Functions of microRNAs in Drosophila development.

Control of mRNA translation and degradation has been shown to be key in the development of complex organisms. The core mRNA degradation machinery is highly conserved in eukaryotes and relies on processive degradation enzymes gaining access to the mRNA. Control of mRNA stability in eukaryotes is also intimately linked to the regulation of translation. A key question in the control of mRNA turnover concerns the mechanisms whereby particular mRNAs are specifically degraded in response to cellular factors. Recently, microRNAs have been shown to bind specifically to mRNAs and regulate their expression via repression of translation and/or degradation. To understand the molecular mechanisms during microRNA repression of mRNAs, it is necessary to identify their biologically relevant targets. However, computational methods have so far proved unreliable, therefore verification of biologically important targets at present requires experimental analysis. The present review aims to outline the mechanisms of mRNA degradation and then focus on the role of microRNAs as factors affecting particular Drosophila developmental processes via their post-transcriptional effects on mRNA degradation and translation. Examples of experimentally verified targets of microRNAs in Drosophila are summarized.

Drosophila 5'-->3'-exoribonuclease Pacman.

Publisher Summary This chapter concentrates on the methods used to express a Drosophila recombinat 5′ → 3′-exoribonuclease, purify the protein, and analyze its activity in vitro . Analysis of early development in Drosophila has shown that RNA localization, control of translation, and mRNA stability are intimately linked. Generally, translational repression leads to degradation of an RNA, and failure of an RNA to localize correctly also leads to its degradation. Work on the yeast Saccharomyces cerevisiae has identified many ribonucleases and associated factors that control mRNA decay, RNA splicing, and rRNA processing. In yeast, it has been shown that 3′ → 5′ degradation/processing of RNA requires the exosome, which is a complex of at least 10 proteins. Degradation of RNA in a 5′ → 3′ direction occurs by initial decapping of the mRNA, followed by 5′ → 3′ degradation of the RNA by Xrn1p. The two decapping proteins (Dcp1p and Dcp2p) and Xrnlp have been shown to be complexed to seven Lsm proteins, which are likely to form a ring encircling the RNA. To understand the role of RNA stability in development, a number of approaches can be used. Once the genes encoding particular ribonucleases or associated factors have been identified, then expression of the RNA during development can be determined. These techniques have been used to show that the 5′ → 3′-exoribonuclease Pacman is differentially expressed during development.

The roles of miRNAs in wing imaginal disc development in Drosophila

During development, it is essential for gene expression to occur in a very precise spatial and temporal manner. There are many levels at which regulation of gene expression can occur, and recent evidence demonstrates the importance of mRNA stability in governing the amount of mRNA that can be translated into functional protein. One of the most important discoveries in this field has been miRNAs (microRNAs) and their function in targeting specific mRNAs for repression. The wing imaginal discs of Drosophila are an excellent model system to study the roles of miRNAs during development and illustrate their importance in gene regulation. This review aims at discussing the developmental processes where control of gene expression by miRNAs is required, together with the known mechanisms of this regulation. These developmental processes include Hox gene regulation, developmental timing, growth control, specification of SOPs (sensory organ precursors) and the regulation of signalling pathways.

RNA-seq reveals post-transcriptional regulation of Drosophila insulin-like peptide dilp8 and the neuropeptide-like precursor Nplp2 by the exoribonuclease Pacman/XRN1

Ribonucleases are critically important in many cellular and developmental processes and defects in their expression are associated with human disease. Pacman/XRN1 is a highly conserved cytoplasmic exoribonuclease which degrades RNAs in a 5′-3′ direction. In Drosophila, null mutations in pacman result in small imaginal discs, a delay in onset of pupariation and lethality during the early pupal stage. In this paper, we have used RNA-seq in a genome-wide search for mRNAs misregulated in pacman null wing imaginal discs. Only 4.2% of genes are misregulated ±>2-fold in pacman null mutants compared to controls, in line with previous work showing that Pacman has specificity for particular mRNAs. Further analysis of the most upregulated mRNAs showed that Pacman post-transcriptionally regulates the expression of the secreted insulin-like peptide Dilp8. Dilp8 is related to human IGF-1, and has been shown to coordinate tissue growth with developmental timing in Drosophila. The increased expression of Dilp8 is consistent with the developmental delay seen in pacman null mutants. Our analysis, together with our previous results, show that the normal role of this exoribonuclease in imaginal discs is to suppress the expression of transcripts that are crucial in apoptosis and growth control during normal development.