Interplay of viral miRNAs and host mRNAs and proteins

Recent experiments indicate that several viruses may encode microRNAs (miRNAs) in cells. Such RNAs may interfere with the host mRNAs and proteins. We present a kinetic analysis of this interplay. In our treatment, the viral miRNA is considered to be able to associate with the host mRNA with subsequent degradation. This process may result in a decline of the mRNA population and also in a decline of the population of the protein encoded by this mRNA. With these ingredients, we first show the types of the corresponding steady-state kinetics in the cases of positive and negative regulation of the miRNA synthesis by the protein. In addition, we scrutinize the situation when the protein regulates the virion replication or, in other words, provides a feedback for the replication. For the negative feedback, the replication rate is found to increase with increasing the intracellular virion population. For the positive feedback, the replication rate first increases and then drops. These features may determine the stability of steady states.

Positive and negative cis-regulatory elements directing postfertilization maternal mRNA translational control in mouse embryos

Mechanisms providing for temporally complex patterns of maternal mRNA translation after fertilization are poorly understood. We employed bioinformatics analysis to compare populations of mRNAs enriched specifically on polysomes at the metaphase II (MII) stage oocyte and late one-cell stages and a detailed deletion/truncation series to identify elements that regulate translation. We used the Bag4 3′ untranslated region (UTR) as a model. Bioinformatics analysis revealed one conserved motif, subsequently confirmed by functional studies to be a key translation repressor element. The deletion/truncation studies revealed additional regulatory motifs, most notably a strong translation activator element of <30 nt. Analysis of mRNA secondary structure suggests that secondary structure plays a key role in translation repression. Additional bioinformatics analysis of the regulated mRNA population revealed a diverse collection of regulatory motifs found in small numbers of mRNAs, highlighting a high degree of sequence diversity and combinatorial complexity in the overall control of the maternal mRNA population. We conclude that translational control after fertilization is driven primarily by negative regulatory mechanisms opposing strong translational activators, with stage-specific release of the inhibitory influences to permit recruitment. The combination of bioinformatics analysis and deletion/truncation studies provides the necessary approach for dissecting postfertilization translation regulatory mechanisms.

EFFECT OF NON-CODING RNA ON BISTABILITY AND OSCILLATIONS IN THE mRNA-PROTEIN INTERPLAY

The feedbacks between the mRNA and protein synthesis may result in kinetic bistability and oscillations. Two generic models predicting bistability include, respectively, a gene with positive regulation of the mRNA production by protein and two genes with mutual suppression of the mRNA production due to negative regulation of the gene transcription by protein. The simplest model predicting oscillations describes a gene with negative regulation of the mRNA production by protein formed via mRNA translation and a few steps of conversion. We complement these models by the steps of non-coding RNA (ncRNA) formation and ncRNA-mRNA association and degradation. With this extension, the bistability can often be observed as well. Without and with ncRNA, the biochemistry behind the steady states may be different. In the latter case, for example, ncRNA may control the mRNA population in the situations when this population is relatively small, and one can observe a switch in the mRNA, protein and ncRNA populations. Our analysis of oscillatory kinetics of the mRNA-protein interplay shows that with ncRNA the oscillations may be observed in a wider range of parameters and the amplitude of oscillations may be larger.

Influence of cucumber mosaic virus infection on the mRNA population present in the phloem translocation stream of pumpkin plants

The effect of cucumber mosaic virus (CMV) infection on the phloem sap mRNA population was investigated in pumpkin Cucurbita maxima Duch. cv. Big Max, through analysis of a suppressive subtractive hybridisation (SSH) library. Analysis of the infected phloem library identified 91 highly diverse mRNA species, including enzymes involved in general metabolism, transcription factors and signalling agents. Our analysis indicated that, quantitatively, the effect of CMV infection on the composition of the phloem sap transcriptome was minor in nature. Virtual northern analysis was used to confirm the specific upregulation of these transcripts in the phloem of CMV-infected plants. In silico northern analysis also confirmed that none of the transcripts identified in the SSH library was contained in the population of mRNA species present in the phloem sap of healthy plants. Induction levels ranged from low to high and in situ hybridisation studies showed that transcripts displayed a range of accumulation patterns. Collectively, our findings suggest that plants have evolved a highly robust mechanism for the exchange of information macromolecules between the companion cell (CC) and the sieve tube system. Production of viral movement protein (MP) in the CC is not sufficient for the indiscriminate transport of mRNA into the sieve element. Our findings are discussed in the context of symptom development and likely strong selection pressure, on the viral genome, to encode for a MP that does not adversely interfere with the phloem long-distance trafficking system.

Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display

ABSTRACT The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacteriumenvironmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.

Expression Profile of Active Genes in Granulocytes

A number of genes active in granulocytes have been intensively studied as to the function of their products and their expression controls. However, the intensities and relative order of these gene activities have not been studied. This report describes an expression profile of 748 different species of active genes in human peripheral granulocytes obtained by analyzing a 3′-directed cDNA library that faithfully represents the mRNA population in the source cells. A significant fraction (20.3% of the total) of the expressed genes in granulocytes consisted of nuclear proteins such as DNA binding proteins, of secretory proteins such as cytokines, and of membrane proteins such as major histocompatibility complex (MHC) proteins and receptors. By comparing this expression profile with 11 profiles similarly obtained with unrelated human cells/tissues, we discovered 10 novel genes that are likely to act specifically in granulocytes. Comparison of this expression profile with that obtained with granulocytoids widely used as a granulocyte model by inducing a cultured promyelocytic leukemia cell line HL60 showed similarities and dissimilarities of gene expressions. © 1998 by The American Society of Hematology.