Macrophage secretion of miR-106b-5p causes renin-dependent hypertension.

Myeloid cells are known mediators of hypertension, but their role in initiating renin-induced hypertension has not been studied. Vitamin D deficiency causes pro-inflammatory macrophage infiltration in metabolic tissues and is linked to renin-mediated hypertension. We tested the hypothesis that impaired vitamin D signaling in macrophages causes hypertension using conditional knockout of the myeloid vitamin D receptor in mice (KODMAC). These mice develop renin-dependent hypertension due to macrophage infiltration of the vasculature and direct activation of renal juxtaglomerular (JG) cell renin production. Induction of endoplasmic reticulum stress in knockout macrophages increases miR-106b-5p secretion, which stimulates JG cell renin production via repression of transcription factors E2f1 and Pde3b. Moreover, in wild-type recipient mice of KODMAC/miR106b-/- bone marrow, knockout of miR-106b-5p prevents the hypertension and JG cell renin production induced by KODMAC macrophages, suggesting myeloid-specific, miR-106b-5p-dependent effects. These findings confirm macrophage miR-106b-5p secretion from impaired vitamin D receptor signaling causes inflammation-induced hypertension.

PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae.

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.

A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf.

Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to various extracellular stimuli in many different cell types. Such regulation of MAPK results from sequential activation of a series of protein kinases. The kinases that phosphorylate MAPKs, the MAP kinase kinases (MEKs) are also activated by phosphorylation. MEKs are related in sequence to the yeast protein kinases Byr1 (from Schizosaccharomyces pombe) and Ste7 (from Saccharomyces cerevisiae), which function in the pheromone-induced signaling pathway that results in mating. Byr1 and Ste7 are in turn regulated by the protein kinases Byr2 and Ste11. The amino acid sequence of the mouse homolog of Byr2 and Ste11, denoted MEKK (MEK kinase), was elucidated from a complementary DNA sequence encoding a protein of 672 amino acid residues (73 kilodaltons). MEKK was expressed in all mouse tissues tested, and it phosphorylated and activated MEK. Phosphorylation and activation of MEK by MEKK was independent of Raf, a growth factor-regulated protein kinase that also phosphorylates MEK. Thus, MEKK and Raf converge at MEK in the protein kinase network mediating the activation of MAPKs by hormones, growth factors, and neurotransmitters.

Diversity in function and regulation of MAP kinase pathways.

Eukaryotic cells from yeast to humans use sequential protein kinase reactions to regulate complex cellular functions. Equivalent protein kinases in different pathways have significant sequence homologies; however, little crossover in phosphorylation of substrates between pathways normally occurs. Assembly of kinase complexes and discrimination of substrates provide the selectivity of sequential protein kinase pathways to regulate such diverse cellular functions as osmoregulation, cell-wall biosynthesis, growth and differentiation.

Postnatal induction and localization of R7BP, a membrane-anchoring protein for regulator of G protein signaling 7 family-Gbeta5 complexes in brain.

Members of the regulator of G protein signaling 7 (RGS7) (R7) family and Gbeta5 form obligate heterodimers that are expressed predominantly in the nervous system. R7-Gbeta5 heterodimers are GTPase-activating proteins (GAPs) specific for Gi/o-class Galpha subunits, which mediate phototransduction in retina and the action of many modulatory G protein-coupled receptors (GPCRs) in brain. Here we have focused on the R7-family binding protein (R7BP), a recently identified palmitoylated protein that can bind R7-Gbeta5 complexes and is hypothesized to control the intracellular localization and function of the resultant heterotrimeric complexes. We show that: 1) R7-Gbeta5 complexes are obligate binding partners for R7BP in brain because they co-immunoprecipitate and exhibit similar expression patterns. Furthermore, R7BP and R7 protein accumulation in vivo requires Gbeta5. 2) Expression of R7BP in Neuro2A cells at levels approximating those in brain recruits endogenous RGS7-Gbeta5 complexes to the plasma membrane. 3) R7BP immunoreactivity in brain concentrates in neuronal soma, dendrites, spines or unmyelinated axons, and is absent or low in glia, myelinated axons, or axon terminals. 4) RGS7-Gbeta5-R7BP complexes in brain extracts associate inefficiently with detergent-resistant lipid raft fractions with or without G protein activation. 5) R7BP and Gbeta5 protein levels are upregulated strikingly during the first 2-3 weeks of postnatal brain development. Accordingly, we suggest that R7-Gbeta5-R7BP complexes in the mouse or rat could regulate signaling by modulatory Gi/o-coupled GPCRs in the developing and adult nervous systems.

G-protein-coupled receptors function as oligomers in vivo.

Hormones, sensory stimuli, neurotransmitters and chemokines signal by activating G-protein-coupled receptors (GPCRs) [1]. Although GPCRs are thought to function as monomers, they can form SDS-resistant dimers, and coexpression of two non-functional or related GPCRs can result in rescue of activity or modification of function [2-10]. Furthermore, dimerization of peptides corresponding to the third cytoplasmic loops of GPCRs increases their potency as activators of G proteins in vitro [11], and peptide inhibitors of dimerization diminish beta(2)-adrenergic receptor signaling [3]. Nevertheless, it is not known whether GPCRs exist as monomers or oligomers in intact cells and membranes, whether agonist binding regulates monomer-oligomer equilibrium, or whether oligomerization governs GPCR function. Here, we report that the alpha-factor receptor, a GPCR that is the product of the STE2 gene in the yeast Saccharomyces cerevisiae, is oligomeric in intact cells and membranes. Coexpression of receptors tagged with the cyan or yellow fluorescent proteins (CFP or YFP) resulted in efficient fluorescence resonance energy transfer (FRET) due to stable association rather than collisional interaction. Monomer-oligomer equilibrium was unaffected by binding of agonist, antagonist, or G protein heterotrimers. Oligomerization was further demonstrated by rescuing endocytosis-defective receptors with coexpressed wild-type receptors. Dominant-interfering receptor mutants inhibited signaling by interacting with wild-type receptors rather than by sequestering G protein heterotrimers. We suggest that oligomerization is likely to govern GPCR signaling and regulation.

Actin cytoskeleton organization regulated by the PAK family of protein kinases.

Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton.

RGS4 causes increased mortality and reduced cardiac hypertrophy in response to pressure overload.

RGS family members are GTPase-activating proteins (GAPs) for heterotrimeric G proteins. There is evidence that altered RGS gene expression may contribute to the pathogenesis of cardiac hypertrophy and failure. We investigated the ability of RGS4 to modulate cardiac physiology using a transgenic mouse model. Overexpression of RGS4 in postnatal ventricular tissue did not affect cardiac morphology or basal cardiac function, but markedly compromised the ability of the heart to adapt to transverse aortic constriction (TAC). In contrast to wild-type mice, the transgenic animals developed significantly reduced ventricular hypertrophy in response to pressure overload and also did not exhibit induction of the cardiac "fetal" gene program. TAC of the transgenic mice caused a rapid decompensation in most animals characterized by left ventricular dilatation, depressed systolic function, and increased postoperative mortality when compared with nontransgenic littermates. These results implicate RGS proteins as a crucial component of the signaling pathway involved in both the cardiac response to acute ventricular pressure overload and the cardiac hypertrophic program.

The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization.

To identify functional domains of G-protein-coupled receptors that control pathway activation, ligand discrimination, and receptor regulation, we have used as a model the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. From a collection of random mutations introduced in the region coding for the third cytoplasmic loop of Ste2p, six ste2sst alleles were identified by genetic screening methods that increased alpha-factor sensitivity 2.5- to 15-fold. The phenotypic effects of ste2sst and sst2 mutations were not additive, consistent with models in which the third cytoplasmic loop of the alpha-factor receptor and the regulatory protein Sst2p control related aspects of pheromone response and/or desensitization. Four ste2sst mutations did not dramatically alter cell surface expression or agonist binding affinity of the receptor; however, they did permit detectable responses to an alpha-factor antagonist. One ste2sst allele increased receptor binding affinity for alpha-factor and elicited stronger responses to antagonist. Results of competition binding experiments indicated that wild-type and representative mutant receptors bound antagonist with similar affinities. The antagonist-responsive phenotypes caused by ste2sst alleles were therefore due to defects in the ability of receptors to discriminate between agonist and antagonist peptides. One ste2sst mutation caused rapid, ligand-independent internalization of the receptor. These results demonstrate that the third cytoplasmic loop of the alpha-factor receptor is a multifunctional regulatory domain that controls pathway activation and/or desensitization and influences the processes of receptor activation, ligand discrimination, and internalization.

Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.

Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.

Mechanisms governing the activation and trafficking of yeast G protein-coupled receptors.

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent "precoupling" of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

Dual lipid modification motifs in G(alpha) and G(gamma) subunits are required for full activity of the pheromone response pathway in Saccharomyces cerevisiae.

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein gamma subunit (Ste18p) is unusual among G(gamma) subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G(gamma) subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G(betagamma) after receptor-stimulated release from G(alpha). The G protein alpha subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.

Translational control of phage f1 gene expression by differential activities of the gene V, VII, IX and VIII initiation sites.

Phage-specific transcription and subsequent RNA processing in Escherichia coli infected with the filamentous phage (f1, M13, fd) generate a pool of abundant and relatively long-lived phage mRNA species encoding the four adjacent genes V, VII, IX and VIII. Yet the products of gene V and gene VIII are synthesized at much higher levels than the gene VII and gene IX proteins. To ask if the translational initiation sites heading these genes show corresponding differences in activity and/or functional properties, we have purified a number of the phage mRNAs from cells infected with f1 and examined them in in vitro initiation reactions. The ribosome binding patterns obtained for the phage mRNA species and for smaller defined RNA fragments containing selected initiator regions reveal a large range in apparent ribosome binding strengths. The gene V and gene VIII sites are recognized efficiently in each mRNA species in which they are present. Gene IX site activity appears to be limited by local mRNA structure: the site has undetectable or low ribosome binding activity in all of the phage mRNA species, but is at least tenfold more active if the RNA sequences required to form a potential hairpin stem-and-loop 15 nucleotides upstream from the initiator AUG have been removed. The gene VII site shows no evidence of interaction with ribosomes in any phage mRNA or RNA fragment tested. The same striking differences in initiation activity were observed in vivo by cloning small f1 DNA fragments containing gene V or gene VII initiation site sequences to drive beta-galactosidase synthesis. High levels of a gene V-beta-galactosidase fusion protein are initiated at the V site, but no detectable synthesis occurs from the VII site. If the VII site is preceded by all of the information encoding the upstream gene V, however, modest amounts of a fusion protein initiated at the VII site are produced. The overall results, in accord with the observed yields of proteins in the phage-infected cell, provide strong evidence that the properties of these translational initiation sites determine in a significant way the differential expression of phage f1 genes V, VII, IX and VIII.

Control of adaptation to mating pheromone by G protein beta subunits of Saccharomyces cerevisiae.

The STE4 gene of the yeast Saccharomyces cerevisiae encodes the beta subunit of a heterotrimeric G protein that mediates response to mating pheromones and influences recovery from pheromone-induced growth arrest. To explore how G beta subunits regulate response and recovery (adaptation), we isolated and characterized signaling-defective STE4 alleles (STE4sd). STE4sd mutations resulted in amino acid substitutions in the N-terminal region of Ste4p, proximal to the first of seven repeat units conserved in G protein beta subunits. Genetic tests indicated that STE4sd mutations disrupted functions of Ste4p required for inducing pheromone responses. Wild-type cells that overexpressed STE4sd alleles displayed apparently normal initial responses to pheromone as judged by quantitative mating, G1 arrest and transcriptional assays. However, after undergoing initial G1 arrest, wild-type cells overexpressing STE4sd alleles recovered more quickly from division arrest, suggestive of a hyperadaptive phenotype. Because hyperadaptation occurred when STE4sd alleles were overexpressed in cells lacking Sst1p (Bar1p), Sst2p or the C-terminal domain of the alpha-factor receptor, this phenotype did not involve three principal modes of adaptation in yeast. However, hyperadaptation was abolished when STE4sd mutations were combined in cis with a deletion that removes a segment of Ste4p (residues 310-346) previously implicated in adaptation to pheromone. These results indicate that G beta subunits possess two independent activities, one required for triggering pheromone response and another that promotes adaptation. Potential models for G beta subunit-mediated adaptation are discussed.

Mot3, a Zn finger transcription factor that modulates gene expression and attenuates mating pheromone signaling in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein beta subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5' flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.

Phage fl mRNA processing in Escherichia coli: search for the upstream products of endonuclease cleavage, requirement for the product of the altered mRNA stability (ams) locus.

In Escherichia coli infected with the filamentous phage f1, a number of the polycistronic phage mRNA species are generated through post-transcriptional processing by host nuclease activity. In this paper we review experimental evidence assessing whether known RNases are involved in mediating these processing events, and we use S1 nuclease mapping methods to visualize putative upstream products of endonuclease cleavage. By examining f1 processing in a phage-infected host bearing a temperature-sensitive allele of the altered message stability locus (ams), we show that production of the major processed species requires a component of the host cell which functions in the messenger RNA decay process.

Beta and gamma subunits of a yeast guanine nucleotide-binding protein are not essential for membrane association of the alpha subunit but are required for receptor coupling.

Conditions were devised to demonstrate GTP-regulated coupling between the yeast STE2-encoded receptor and its cognate guanine nucleotide-binding protein (G protein). Treatment of partially purified membranes with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) converted the receptor from a high-affinity state (Kd = 17 nM) to a much lower affinity state (Kd approximately 150 nM), as judged by three independent criteria: rate of ligand (alpha-factor) dissociation, equilibrium binding, and antagonist competition. Expression of STE2 from the GAL1 promoter in MATa/MAT alpha diploids, which do not express GPA1 (encoding G protein alpha subunit, G alpha), STE4 (encoding G protein beta subunit, G beta), and STE18 (encoding G protein gamma subunit, G gamma) but do express another G protein alpha subunit (product of GPA2), yielded a single class of low-affinity receptors that were GTP[gamma-S]-insensitive, indicating that STE2 gene product cannot couple productively with other G proteins, even in the absence of competition by its cognate G protein. By using gpa1, STE4, and ste18 mutations, it was found that all three G protein subunits were required for functional coupling, as judged by the absence of high-affinity receptors when any of the three gene products was altered. This finding demonstrates that G beta and G gamma subunits are essential for formation of a productive complex between a G alpha subunit and its corresponding receptor. Wild-type STE4 and STE18 gene products were not essential for membrane localization of the GPA1 gene product, as indicated by cell fractionation and immunological analyses, suggesting that G beta and G gamma subunits interact with the receptor or make the G alpha subunit competent to associate correctly with the receptor, or both.

Recognition and cleavage signals for mRNA processing lie within local domains of the phage f1 RNA precursors.

In Escherichia coli infected with the filamentous phage f1, a number of the abundant phage mRNAs, species C-G, are products of post-transcriptional processing. The approach of cloning the phage sequences likely to include the processing signals in a plasmid under transcriptional control of the lambda PL promoter (Blumer, K. J., and Steege, D. A. (1984) Nucleic Acids Res. 12, 1847-1861) was extended to additional sites to show that processing at all five major sites is mediated by nuclease activity encoded by the bacterial genome. Primer extension methods were used to map more accurately in the f1 DNA sequence the 5' end points of the processed RNAs. The DNA segments that encode the mRNA processing signals were delimited from parallel series of 5'-3' and 3'-5' deletions made into the regions in which the RNA 5' ends map. For each deletion variant, in vivo f1 mRNA processing activity was assessed by primer extension and S1 nuclease mapping methods. The data indicate that the processing signals are comprised of relatively local regions near the point of RNA cleavage. Whereas cleavage occurs at the 5' border of the sequences that comprise the D, E, and F processing sites and thereby places most of the recognition information in the mature or product portion of the precursor, it occurs more centrally within the region comprising the C site. The C and D sites function independently as substrates for cleavage, with the necessary information contained in regions of 70 and 90 nucleotides, respectively. Cleavage at the E site appears to require a region of 130 nucleotides which completely contains the F site. From the effects on processing activity of deleting sequences in this region, the overlapping E and F processing sites appear to consist functionally of two subdomains. Each has a cleavage site at its immediate 5' end which can be substituted by foreign sequences, but both utilize a common recognition domain downstream from the point of strand scission.

mRNA processing in Escherichia coli: an activity encoded by the host processes bacteriophage f1 mRNAs.

To examine the regions of the male-specific filamentous bacteriophage f1 genome that include signals for mRNA processing, the 5' endpoints of the major in vivo phage mRNAs have been located in the f1 DNA sequence by S1 nuclease mapping. The 5' ends of the purified mRNAs and additional phage-specific RNAs transiently visible early after infection occur in clusters of T-rich residues within genes that code for three phage proteins. When a 270-nucleotide region encompassing the 5' endpoints of three processed RNAs is transcribed as part of the bacteriophage lambda N mRNA in uninfected female cells, RNA 5' ends identical to ends of the three f1 RNAs are generated from the lambda-f1 precursor. This finding indicates that the mRNA processing activity is encoded by the bacterial host, and that its recognition sites are present in the local regions near the 5' ends which result from RNA cleavage. Several characteristics of f1 mRNA processing events have implications for the differential regulation of adjacent phage genes constrained in the same transcription unit, and may be representative of similar processing events occurring in the bacterial cell.