Tiling microarrays have proven to be a valuable tool for gaining insights into the transcriptomes of microbial organisms grown under various nutritional or stress conditions. Here, we describe the use of such an array, constructed at the level of 20 nt resolution for the Escherichia coli MG1655 genome, to observe genome-wide changes in the steady-state RNA levels in mutants defective in either RNase E or RNase III. The array data were validated by comparison to previously published results for a variety of specific transcripts as well as independent northern analysis of additional mRNAs and sRNAs. In the absence of RNase E, 60% of the annotated coding sequences showed either increases or decreases in their steady-state levels. In contrast, only 12% of the coding sequences were affected in the absence of RNase III. Unexpectedly, many coding sequences showed decreased abundance in the RNase E mutant, while more than half of the annotated sRNAs showed changes in abundance. Furthermore, the steady-state levels of many transcripts showed overlapping effects of both ribonucleases. Data are also presented demonstrating how the arrays were used to identify potential new genes, RNase III cleavage sites and the direct or indirect control of specific biological pathways.

SUMMARY Cellular response to osmotic stress is critical for survival and involves volume control through the regulated transport of osmolytes [1–3]. Organelles may respond similarly to abrupt changes in cytoplasmic osmolarity [4–6]. The plastids of the Arabidopsis thaliana leaf epidermis provide a model system for the study of organellar response to osmotic stress within the context of the cell. An Arabidopsis mutant lacking two plastid-localized homologs of the bacteria mechanosensitive channel MscS (MscS-Like (MSL) 2 and 3) exhibits large round epidermal plastids that lack dynamic extensions known as stromules [7]. This phenotype is present under normal growth conditions and does not require exposure to extracellular osmotic stress. Here, we show that increasing cytoplasmic osmolarity through a genetic lesion known to produce elevated levels of soluble sugars, exogenously providing osmolytes in the growth media, or withholding water rescues the msl2-1 msl3-1 leaf epidermal plastid phenotype, producing plastids that resemble the wild type in shape and size. Furthermore, the epidermal plastids in msl2-1 msl3-1 leaves undergo rapid and reversible volume and shape changes in response to extracellular hypertonic or hypotonic challenges. We conclude that plastids are under hypoosmotic stress during normal plant growth and dynamic response to this stress requires MSL2 and MSL3.

In eukaryotes, box C͞D modification guide RNAs are made by two primary pathways (2, 5, 10, 11). Some are found in the introns of host genes, and the linear box C͞D RNAs are processed from debranched lariats. In other cases, linear box C͞D RNAs are processed from mono-or polycistronic transcripts synthesized from independent promoters. Box C͞D RNAs were found only recently in archaea (12, 13). The pathway by which archaeal box C͞D RNAs are made is unknown. The archaeal box C͞D RNAs are located primarily in intergenic regions of the genome, sometimes overlapping the 3Ј or 5Ј end of flanking ORFs (1, 4, 12, 13). Independent promoters have not been identified.In the course of analyzing a cDNA library of small RNAs from P. furiosus, we observed an interesting incongruence between the linear sequence of some box C͞D RNA clones