Prochlorococcus, an extremely small cyanobacterium that is very abundant in the world's oceans, has a very streamlined genome. On average, these cells have about 2,000 genes and very few regulatory proteins. The limited capability of regulation is thought to be a result of selection imposed by a relatively stable environment in combination with a very small genome. Furthermore, only ten non-coding RNAs (ncRNAs), which play crucial regulatory roles in all forms of life, have been described in Prochlorococcus. Most strains also lack the RNA chaperone Hfq, raising the question of how important this mode of regulation is for these cells. To explore this question, we examined the transcription of intergenic regions of Prochlorococcus MED4 cells subjected to a number of different stress conditions: changes in light qualities and quantities, phage infection, or phosphorus starvation. Analysis of Affymetrix microarray expression data from intergenic regions revealed 276 novel transcriptional units. Among these were 12 new ncRNAs, 24 antisense RNAs (asRNAs), as well as 113 short mRNAs. Two additional ncRNAs were identified by homology, and all 14 new ncRNAs were independently verified by Northern hybridization and 5′RACE. Unlike its reduced suite of regulatory proteins, the number of ncRNAs relative to genome size in Prochlorococcus is comparable to that found in other bacteria, suggesting that RNA regulators likely play a major role in regulation in this group. Moreover, the ncRNAs are concentrated in previously identified genomic islands, which carry genes of significance to the ecology of this organism, many of which are not of cyanobacterial origin. Expression profiles of some of these ncRNAs suggest involvement in light stress adaptation and/or the response to phage infection consistent with their location in the hypervariable genomic islands.

SummaryMicro-RNAs (miRNAs) are one class of small non-coding RNAs that have important regulatory roles in higher plants. Much less is known about their prevalence and function in lower land plants. Previously we cloned 100 non-structural small RNAs from the moss Physcomitrella patens but could annotate only 11 as miRNAs. To identify additional moss miRNAs among cloned small RNAs we have analyzed their genomic sequences for a characteristic miRNA precursor-like structure. This analysis revealed 19 new moss miRNAs that are predicted to be encoded by 22 putative foldbacks. Northern blot analysis confirmed the expression of 14 new miRNA representatives. Half of these were gametophore specific, the rest were detected at low levels in the protonema. We predicted 12 genes as targets of nine new miRNAs. Three of these show homology to transcription factors and the others appear to play roles in diverse physiological processes including light and cytokine signaling, which have not to date been shown to be regulated by a miRNA in flowering plants. Four target genes, which show homology to ATN1-like protein kinase, NAC transcription factors and a cytokinin receptor, have been validated by miRNA-mediated mRNA cleavage. In addition, our analysis revealed that seven small RNAs represent miRNA* and three represent intermediates of pre-miRNA processing, providing evidence for specific DICER-like cleavage steps during miRNA biogenesis in moss. Our findings suggest that miRNAs are common in mosses and set the stage for the elucidation of their varied biological functions.

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Retrograde signaling is a pathway of communication from mitochondria and plastids to the nucleus in the context of cell differentiation, development, and stress response. In Chlamydomonas reinhardtii, the tetrapyrroles magnesium-protoporphyrin IX and heme are only synthesized within the chloroplast, and they have been implicated in the retrograde control of nuclear gene expression in this unicellular green alga. Feeding the two tetrapyrroles to Chlamydomonas cultures was previously shown to transiently induce five nuclear genes, three of which encode the heat shock proteins HSP70A, HSP70B, and HSP70E. In contrast, controversial results exist on the possible role of magnesium-protoporphyrin IX in the repression of genes for lightharvesting proteins in higher plants, raising the question of how important this mode of regulation is. Here, we used genomewide transcriptional profiling to measure the global impact of these tetrapyrroles on gene regulation and the scope of the response. We identified almost 1,000 genes whose expression level changed transiently but significantly. Among them were only a few genes for photosynthetic proteins but several encoding enzymes of the tricarboxylic acid cycle, heme-binding proteins, stress-response proteins, as well as proteins involved in protein folding and degradation. More than 50% of the latter class of genes was also regulated by heat shock. The observed drastic fold changes at the RNA level did not correlate with similar changes in protein concentrations under the tested experimental conditions. Phylogenetic profiling revealed that genes of putative endosymbiontic origin are not overrepresented among the responding genes. This and the transient nature of changes in gene expression suggest a signaling role of both tetrapyrroles as secondary messengers for adaptive responses affecting the entire cell and not only organellar proteins.

In recent years, sRNAs (small non-coding RNAs) have been found to be abundant in eukaryotes and bacteria and have been recognized as a novel class of gene expression regulators. In contrast, much less is known about sRNAs in archaea, except for snoRNAs (small nucleolar RNAs) that are involved in the modification of bases in stable RNAs. Therefore bioinformatic and experimental RNomics approaches were undertaken to search for the presence of sRNAs in the model archaeon Haloferax volcanii, resulting in more than 150 putative sRNA genes being identified. Northern blot analyses were used to study (differential) expression of sRNA genes. Several chromosomal deletion mutants of sRNA genes were generated and compared with the wild-type. It turned out that two sRNAs are essential for growth at low salt concentrations and high temperatures respectively, and one is involved in the regulation of carbon metabolism. Taken together, it could be shown that sRNAs are as abundant in H. volcanii as they are in well-studied bacterial species and that they fulfil important biological roles under specific conditions.