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Regulating the expression of individual miRNAs (microRNAs) is important for cell development and function. The up- or down-regulation of the processing of specific miRNA precursors to the mature active form represents one tool to control miRNA concentration and is mediated by proteins that recognize the terminal loop of the RNA precursors. Terminal loop recognition is achieved by the combined action of several RNA-binding domains. The proteins can then regulate the processing by recruiting RNA enzymes, changing the RNA structure and preventing or enhancing the accessibility and processing activity of the core processing complexes. The present review focuses on how terminal loop-binding proteins recognize their RNA targets and mediate their regulatory function(s), and highlights how terminal loop-mediated regulation relates to the broader regulation of mRNA metabolism.

Localization of the miRNA-induced silencing complex to GW/P bodies by GW220/TNGW1 may regulate the fate of target mRNAs., The microRNA (miRNA)-induced silencing complex (miRISC) controls gene expression by a posttranscriptional mechanism involving translational repression and/or promoting messenger RNA (mRNA) deadenylation and degradation. The GW182/TNRC6 (GW) family proteins are core components of the miRISC and are essential for miRNA function. We show that mammalian GW proteins have distinctive functions in the miRNA pathway, with GW220/TNGW1 being essential for the formation of GW/P bodies containing the miRISC. miRISC aggregation and formation of GW/P bodies sequestered and stabilized translationally repressed target mRNA. Depletion of GW220 led to the loss of GW/P bodies and destabilization of miRNA-targeted mRNA. These findings support a model in which the cellular localization of the miRISC regulates the fate of the target mRNA.

PMCID: PMC1276709, Phage ø29 is a virulent phage of Bacillus subtilis with no known lysogenic cycle. Indeed, lysis occurs rapidly following infection of vegetative cells. Here, we show that 29 possesses a powerful strategy that enables it to adapt its infection strategy to the physiological conditions of the infected host to optimize its survival and proliferation. Thus, the lytic cycle is suppressed when the infected cell has initiated the process of sporulation and the infecting phage genome is directed into the highly resistant spore to remain dormant until germination of the spore. We have also identified two host-encoded factors that are key players in this adaptive infection strategy. We present evidence that chromosome segregation protein Spo0J is involved in spore entrapment of the infected ø29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses ø29 development by repressing the main early ø29 promoters via different and novel mechanisms and also by preventing activation of the single late ø29 promoter., This investigation was supported by grants 2RO1 GM27242-24 from the National Institutes of Health and BMC2002-03818 from the Spanish Ministry of Science and Technology to MS and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular 'Severo Ochoa'. VC is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology, which also supported WJJM by means of the 'Ramón y Cajal' program.

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage φ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the φ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the φ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane., Spanish Ministry of Education and Science Grants BFU2005-00733 (to M.S.) and BFU2005-01878 (to W.J.J.M.), Spanish Ministry of Science and Innovation Grant BFU2008-00215 (to M.S.), Spanish Ministry of Education and Science Grant Consolider-Ingenio 2010 24717 (to M.S.) and by an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘‘Severo Ochoa

The φ29 family of phages is divided in three groups. Members of groups 1 and 2 infect the spore-forming bacterium Bacillus subtilis. Previous studies showed that group 1 phage φ29 adapts its infection strategy to the physiological state of the host. Thus, the lytic cycle of φ29 is suppressed when cells are infected during the early stages of sporulation and the infecting genome becomes trapped into the spore. A major element of this adaptive strategy is a very sensitive response to the host-encoded Spo0A protein, the key regulator for sporulation activation, which is directly responsible for suppression of φ29 development. Here we analysed if this adaptation is conserved in phage Nf belonging to group 2. The results obtained show that although Nf also possesses the alternative infection strategy, it is clearly less sensitive to Spo0A-mediated suppression than φ29. Sequence determination of the Nf genome revealed striking differences in the number of Spo0A binding site sequences. The results provide evidence that the life style of two highly related phages is distinctly tuned by differences in binding sites for a host-encoded regulatory protein, being a good example of how viruses have evolved to optimally exploit features of their host., This investigation was supported by Grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to M.S. and W.J.J.M. respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. V.C. is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology.

Members of groups 1 (e.g. ϕ29) and 2 (e.g. Nf) of the ϕ29 family of phages infect the spore forming bacterium Bacillus subtilis. Although classified as lytic phages, the lytic cycle of ϕ29 can be suppressed and its genome can become entrapped into the B. subtilis spore. This constitutes an alternative infection strategy that depends on the presence of binding sites for the host-encoded protein Spo0A in the ϕ29 genome. Binding of Spo0A to these sites represses ϕ29 transcription and prevents initiation of DNA replication. Although the Nf genome can also become trapped into B. subtilis spores, in vivo studies showed that its lytic cycle is less susceptible to spo0A-mediated suppression than that of ϕ29. Here we have analysed the molecular mechanism underlying this difference showing that Spo0A differently affects transcription and replication initiation of the genomes of these phages. Thus, whereas Spo0A represses all three main early promoters of ϕ29, it only represses one out of the three equivalent early promoters of Nf. In addition, contrary to ϕ29, Spo0A does not prevent the in vitro initiation of Nf DNA replication. Altogether, the differences in Spo0A-mediated regulation of transcription and replication between ϕ29 and Nf explain their different behaviours in vivo., Spanish Ministry of Education and Science (BFU2005-00733 to M.S., BFU2005-01878 to W.J.J.M.); Spanish Ministry of Science and Innovation (BFU2008-00215/BMC to M.S.); institutional grant—Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’; predoctoral fellowship from the Spanish Ministry of Education and Science (to V.C.L.L.). Funding for open access charge: BFU2008-00215/BMC. Conflict of interest statement. None declared.

The host of the lytic bacteriophage φ29 is the spore-forming bacterium Bacillus subtilis. When infection occurs during early stages of sporulation, however, φ29 development is suppressed and the infecting phage genome becomes trapped into the developing spore. Recently, we have shown that Spo0A, the key transcriptional regulator for entry into sporulation, is directly responsible for suppression of the lytic φ29 cycle in cells having initiated sporulation. Surprisingly, we found that φ29 development is suppressed in a subpopulation of logarithmically growing culture and that spo0A is heterogeneously expressed during this growth stage. Furthermore, we showed that kinC and, to a minor extent, kinD, are responsible for heterogeneous expression levels of spo0A during logarithmical growth that are below the threshold to activate sporulation, but sufficient for suppression of the lytic cycle of φ29. Whereas spo0A was known to be heterogeneously expressed during the early stages of sporulation, our findings show that this also occurs during logarithmical growth. These insights are likely to have important consequences, not only for the life cycle of φ29, but also for B. subtilis developmental processes., This investigation was supported by Grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to M.S. and W.J.J.M. respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. V.C. is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology.

The transcription factor Spo0A is a master regulator for entry into sporulation in Bacillus subtilis and also regulates expression of the virulent B. subtilis phage 29. Here, we describe a novel function for Spo0A, being an inhibitor of DNA replication of both, the 29 genome and the B. subtilis chromosome. Binding of Spo0A near the 29 DNA ends, constituting the two origins of replication of the linear 29 genome, prevents formation of 29 protein p6-nucleoprotein initiation complex resulting in inhibition of 29 DNA replication. At the B. subtilis oriC, binding of Spo0A to specific sequences, which mostly coincide with DnaA-binding sites, prevents open complex formation. Thus, by binding to the origins of replication, Spo0A prevents the initiation step of DNA replication of either genome. The implications of this novel role of Spo0A for phage 29 development and the bacterial chromosome replication during the onset of sporulation are discussed., This investigation was supported by grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to MS and WJJM, respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular 'Severo Ochoa'. VC and DM are holders of predoctoral fellowships from the Spanish Ministry of Science and Technology and the Fondo de Investigación Sanitaria, respectively. WJJM was supported by means of the 'Ramón y Cajal' program from the Spanish Ministry of Science and Technology.