Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.

A major class of bacterial small, noncoding RNAs (sRNAs) acts by base-pairing with mRNAs to alter the translation from and/or stability of the transcript. Our laboratory has shown that Hfq, the chaperone that mediates the interaction of many sRNAs with their targets, is required for the virulence of the enteropathogen Yersinia pseudotuberculosis. This finding suggests that sRNAs play a critical role in the regulation of virulence in this pathogen, but these sRNAs are not known. Using a deep sequencing approach, we identified the global set of sRNAs expressed in vitro by Y. pseudotuberculosis. Sequencing of RNA libraries from bacteria grown at 26°C and 37°C resulted in the identification of 150 unannotated sRNAs. The majority of these sRNAs are Yersinia specific, without orthologs in either Escherichia coli or Salmonella typhimurium. Six sRNAs are Y. pseudotuberculosis specific and are absent from the genome of the closely related species Yersinia pestis. We found that the expression of many sRNAs conserved between Y. pseudotuberculosis and Y. pestis differs in both timing and dependence on Hfq, suggesting evolutionary changes in posttranscriptional regulation between these species. Deletion of multiple sRNAs in Y. pseudotuberculosis leads to attenuation of the pathogen in a mouse model of yersiniosis, as does the inactivation in Y. pestis of a conserved, Yersinia-specific sRNA in a mouse model of pneumonic plague. Finally, we determined the regulon controlled by one of these sRNAs, revealing potential virulence determinants in Y. pseudotuberculosis that are regulated in a posttranscriptional manner.Ysr29 | RybB | stress | Illumina-Solexa | 2D-DIGE

In Drosophila melanogaster, the small interfering RNA (siRNA) pathway is triggered by exogenous double-stranded RNA (dsRNA) or upon viral infection. This pathway requires Dicer-2 (Dcr-2) in association with a dsRNA binding protein (dsRBP) called R2D2. A potentially distinct siRNA pathway, which requires Dcr-2 in association with a different dsRBP, called Loquacious (Loqs), is activated by endogenous dsRNA derived from transposons, structured loci and overlapping transcripts. Here, we show that different sources of dsRNA enter a common siR-NA pathway that requires R2D2 and Loqs. R2D2 and loqs mutants show impaired silencing triggered by injection of exogenous dsRNA or by artificial and natural expression of endogenous dsRNA. In addition, we show that these dsRBPs function sequentially and non-redundantly in collaboration with Dcr-2. Loqs is primarily required for dsRNA processing while R2D2 is essential for the subsequent loading of siRNAs into effector Ago-RISC complexes.RNA interference (RNAi) uses small non-coding RNAs to regulate post-transcriptional gene expression. Within somatic animal cells, at least two different species of small RNAs are found: siRNAs and microRNAs (miRNAs) 1 . In Drosophila, the biogenesis and action of miRNAs and siRNAs require distinct machineries 1 . miRNAs originate from nuclear hairpin RNAs that are sequentially processed by two different RNaseIII/dsRBP complexes: Drosha/ Pasha and Dicer-1/Loqs isoform PB (Dcr-1/Loqs-PB), respectively. The resulting mature miRNA is then loaded into the Argonaute protein Ago1. siRNAs originating from exogenous dsRNAs require a different Dicer-dsRBP complex, Dcr-2/R2D2, and are loaded into the Ago2 protein. Exogenous, in this case, refers to dsRNAs that do not undergo nuclear synthesis but originate from cytoplasmic or extra-cellular locales such as experimentally introduced dsRNAs. R2D2 and Loqs perform different biochemical functions with their cognate Dicer partners. Loqs-PB is required for processing of miRNA precursors by Dcr-1 but not for loading of mature miRNAs into Ago1 2 . R2D2 is required for Dcr-2 dependent loading of siRNAs into Ago2 but appears dispensable for processing of exogenous dsRNAs 3,4 .* Corresponding author: Richard Carthew, 847-467-4891, r-carthew@northwestern.edu, Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL. # These authors contributed equally to this work Author contributions: J.T.M. and K.K. conceived and designed the experiments. J.T.M., K.K., P.H.W., T.A. and N.J. performed the experiments. J.T.M, K.K. and R.W.C. analyzed the data. J.T.M. and R.W.C. wrote the paper. Accession numbers:The deep sequencing datasets described in this manuscript are available at NCBI GEO under the accession number GSE18871. Recent studies reported the existence of siRNAs derived from endogenous dsRNAs in C. elegans, Drosophila and mice [5][6][7][8][9][10][11][12][13][14] . In Drosophila and mammals, endogenous siRNAs (endo-siRNAs) are produced from nuclear dsRNAs originating ...

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