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.

Control of mRNA stability in eukaryotes.

mRNA turnover plays a key role in the control of gene expression. Recent work has shown that proteins involved in mRNA turnover are located in multicomponent complexes which are tightly regulated. The control of mRNA stability is also intimately linked with translational processes. This article reviews the pathways and enzymes that control mRNA turnover in eukaryotic cells and discusses their mechanisms of control.

The Drosophila gene twister, an orthologue of the yeast helicase SKI2, is differentially expressed during development.

We have identified and characterized a Drosophila orthologue of SKI2, which, in Saccharomyces cerevisiae, is one of the key components in the cytoplasmic 3'-5' decay of mRNA. The Drosophila orthologue (twister, tst), is expressed as two transcripts which differ in the lengths of their 3'-UTRs, with the smaller transcript being particularly abundant in 0-2 h embryos and the larger transcript reaching its highest levels in 6-8 h embryos. TST protein is expressed in two forms which are differentially expressed in adult tissues and throughout development. Differential expression of TST may modulate activity of the mRNA turnover pathway and could have a major impact on the expression of target RNAs.

Characterization of an SRY-like gene, DSox14, from Drosophila.

We have characterized the DSox14 gene, a new member of the family of transcription factors related to the mammalian sex determining factor, SRY. It contains two exons and the intron is large for Drosophila at 2.8 kb. The encoded protein consists of 691 amino acids (72 kDa) and includes an HMG box domain, which is closely related to the mouse Sox4 DNA binding domain. Expression of the DSox14 HMG box domain in vitro shows that it binds the sequence AACAAT with a K(d) of 190 nM, generating a bend angle of 48.6 degrees. At higher protein concentrations, a second HMG box binds at the recognition sequence, increasing the bend angle by 5 degrees. DSox14 is variably expressed throughout development as three alternative transcripts but not at all during the 1st and 2nd larval instars. The several mRNA transcripts are produced primarily from different transcriptional start sites. Analysis of the expression of DSox14 mRNAs during early development shows that they are maternally contributed at a low level and ubiquitously expressed during embryogenesis. The widespread pattern of expression suggests that DSox14 affects a large number of target genes.

Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster.

In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue (pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)9 which encodes polyglutamine. By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results. Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.

A new role for RNase II in mRNA decay: striking differences between RNase II mutants and similarities with a strain deficient in RNase E.

The effect of Escherichia coli ribonuclease II and polynucleotide phosphorylase was analysed on the degradation of Desulfovibrio vulgaris cytochrome c3 (cyc) mRNA. In the absence of these exoribonucleolytic activities, cyc mRNA was stabilised but the two enzymes had a different role in its decay. Surprisingly, a temperature-sensitive mutation in ribonuclease II gave a degradation pattern similar to what had been observed in the absence of endoribonuclease E activity. In an RNase II deletion mutant this was not observed. We propose and verify a model in which the temperature-sensitive ribonuclease II interferes with the action of ribonuclease E.

Degradation products of the cytochrome c3 mRNA are similar in Desulfovibrio vulgaris Hildenborough and Escherichia coli.

The transcription and mRNA degradation pattern of a cloned Desulfovibrio vulgaris (Dv) Hildenborough cytochrome c3-encoding gene (cyc) was analyzed in detail, both in Escherichia coli and its native species. Transcription in Dv seems to be controlled by the same promoter elements as in E. coli; the transcription start point (tsp) of this Dv gene has been mapped in both species and found to be identical. A major putative transcription terminator was mapped and it was found to be the same in both organisms. Furthermore, the intermediates in cyc mRNA degradation are similar in both bacterial species.

RNase E can inhibit the decay of some degradation intermediates: degradation of Desulfovibrio vulgaris cytochrome c3 mRNA in E coli.

In Escherichia coli, ribonuclease E (RNase E) is a key endonuclease in mRNA decay. We have analysed the role of E coli RNase E on the degradation of a heterologous cytochrome c3 (cyc) mRNA from Desulfovibrio vulgaris Hildenborough. The decay of the cyc transcript in wild-type and mutant E coli cells was followed and the degradation intermediates analysed by Northern blotting and S1 protection analysis. The half-life of total cyc mRNA intermediates was increased in the RNase E mutant. A number of degradation intermediates were stabilised, and new species arose. However, some species decayed faster in the met5 mutant at the non-permissive temperature, suggesting that RNase E might inhibit their degradation. The results indicate that RNase E is involved in cyc mRNA degradation, and, interestingly, decay of certain intermediates could be reduced by this enzyme activity. This may suggest a functional interaction between RNase E and exonucleases, like polynucleotide phosphorylase.

Degradation of maternal string mRNA is controlled by proteins encoded on maternally contributed transcripts.

In Drosophila, maternal string mRNAs are stable for the first few hours of development, but undergo specific timed degradation at the cellularisation stage. To determine whether the proteins that control this degradation are maternally or zygotically transcribed, we have used in situ hybridisation to determine the fate of maternal string transcripts in mutant embryos which lack individual chromosome arms. Our data indicate that maternal string mRNA persists for the normal period in all these mutants. Using alpha-amanitin to inhibit zygotic transcription we have shown that degradation of maternal mRNA is unaffected. Therefore, the proteins required to activate the degradation of string mRNA are encoded on a maternally contributed mRNA. We discuss possible models to explain the degradation pathway.

Compact structures of d(CNG)n oligonucleotides in solution and their possible relevance to fragile X and related human genetic diseases.

We show that oligonucleotides of CNG tracts readily adopt compact DNA structures that move unusually fast on gels. Base composition does not explain this, and non-CNG triplets (including GNC) do not form such structures. Chemical probing and melting experiments suggest that the structures probably are not hairpins. Although both long and short tracts can adopt compact structures, the structure formed by longer tracts is more compact than that formed by shorter ones. We note the possibility that such structures may form in vivo, and be instrumental in normal and/or abnormal function of human genes.

mRNA degradation by processive 3'-5' exoribonucleases in vitro and the implications for prokaryotic mRNA decay in vivo.

Two 3'-5' exoribonucleases, polynucleotide phosphorylase and ribonuclease II play a central role in the degradation of bacterial mRNA to ribonucleotides. Sequences with the potential to form stem-loop structures can stabilize upstream mRNA against 3'-5' exoribonucleolytic attack in vivo by blocking the processive activities of these enzymes. For many mRNA species stem-loop structures appear to provide a very efficient block to decay from the 3' end, such that the rate-determining step for mRNA decay occurs elsewhere in the transcript. We have examined the stalling of 3'-5' exoribonucleases at stem-loop structures in vitro. Although stem-loop structures alone can impede the progress of both enzymes, the duration of stalling at these structures in vitro is insufficient to account for the increased half-lives that they confer on mRNA in vivo. These data suggest that an additional factor, such as a stem-loop binding protein, is required for stabilization of mRNA by stem-loop structures in vivo. The implications for the regulation of mRNA stability are discussed.

Repetitive extragenic palindromic sequences, mRNA stability and gene expression: evolution by gene conversion? A review.

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeats present in up to 1000 copies on the Escherichia coli chromosome. We have shown both in vivo and in vitro that REP sequences can stabilize upstream mRNA by blocking the processive action of 3'----5' exonucleases. In a number of operons, mRNA stabilization by REP sequences plays an important role in the control of gene expression. Furthermore, differential mRNA stability mediated by the REP sequences can be responsible for differential gene expression within polycistronic operons. Despite the key role of REP sequences in mRNA stability and gene expression in a number of operons, several lines of evidence suggest that this is unlikely to be the primary reason for the exceptionally high degree of sequence conservation between REP sequences. Other possible functions for REP sequences are discussed. We propose that REP sequences may be a prokaryotic equivalent of 'selfish DNA' and that gene conversion may play a role in the evolution and maintenance of REP sequences.

Differential mRNA stability controls relative gene expression within a polycistronic operon.

In this paper we demonstrate a role for mRNA stability in controlling relative gene expression within a polycistronic operon. The polycistronic malEFG operon of E. coli contains two REP sequences (highly conserved inverted repeats) within the malE-malF intercistronic region. Deletion of these REP sequences from the chromosomal operon not only destabilizes upstream malE mRNA, but also results in a 9-fold reduction in the synthesis of MalE protein. A single REP sequence seems to be as efficient as the two normally found in this intergenic region at stabilizing translationally active upstream mRNA. The widespread occurrence of REP sequences and other sequences that could potentially stabilize upstream mRNA suggests that this mechanism of control of gene expression may be rather common.

Stabilization of translationally active mRNA by prokaryotic REP sequences.

The REP sequence is a highly conserved inverted repeat that is present in about 25% of all E. coli transcription units. We show that the REP sequence can stabilize upstream RNA, independently of any other sequences, by protection from 3'-5' exonuclease attack. The REP sequence is frequently responsible for the differential stability of different segments of mRNA within an operon. We demonstrate that REP-stabilized mRNA can be translated in vivo and that cloning the REP sequence downstream of a gene can increase protein synthesis. This provides direct evidence that alterations in mRNA stability can play a role in determining bacterial gene expression. The implications of these findings for the mechanisms of mRNA degradation and for the role of RNA stability in the regulation of gene expression are discussed.

Sequence analysis of the pyr-4 (orotidine 5'-P decarboxylase) gene of Neurospora crassa.

The pyr-4 gene of Neurospora crassa encodes orotidine-5' -phosphate decarboxylase, which catalyses the sixth step in the pyrimidine biosynthetic pathway. The complete nucleotide sequence of a 1.8-kb genomic fragment containing the pyr-4 gene has been determined. Using transposon mutagenesis, the coding region has been identified, and the amino acid (aa) sequence deduced. Comparison of the pyr-4 aa sequence with URA3, the equivalent gene of Saccharomyces cerevisiae, showed extensive blocks of homology, with non-homologous sequences between these blocks being generally much longer in Neurospora than in yeast. Computer-predicted protein secondary structure of pyr-4 and URA3 was conserved within equivalent blocks. Upstream sequences of pyr-4 were compared with other sequenced Neurospora genes and possible promoter sequences identified.