Pleckstrin homology domains interact with filamentous actin.

A fraction of Bruton's tyrosine kinase (Btk) co-localizes with actin fibers upon stimulation of mast cells via the high affinity IgE receptor (FcepsilonRI). In this study, a molecular basis of the Btk co-localization with actin fibers is presented. Btk and other Tec family tyrosine kinases have a pleckstrin homology (PH) domain at their N termini. The PH domain is a short peptide module frequently found in signal-transducing proteins and cytoskeletal proteins. Filamentous actin (F-actin) is shown to be a novel ligand for a subset of PH domains, including that of Btk. The actin-binding site was mapped to a 10-residue region of the N-terminal region of Btk. Basic residues in this short stretch are demonstrated to be involved in actin binding. Isolated PH domains induced actin filament bundle formation. Consistent with these observations, Btk binds F-actin in vitro and in vivo. Wild-type Btk protein is in part translocated to the cytoskeleton upon FcepsilonRI cross-linking, whereas Btk containing a mutated PH domain is not. Phosphatidylinositol 3,4, 5-trisphosphate-mediated membrane translocation of Btk was enhanced in cytochalasin D-pretreated, FcepsilonRI-stimulated mast cells. These data indicate that PH domain-mediated F-actin binding plays a role in Btk co-localization with actin filaments.     

Multiple roles of pleckstrin homology domains in phospholipase Cbeta function

Since their discovery almost 10 years ago pleckstrin homology (PH) domains have been identified in a wide variety of proteins. Here, we focus on two proteins whose PH domains play a defined functional role, phospholipase C (PLC)-beta(2) and PLCdelta(1). While the PH domains of both proteins are responsible for membrane targeting, their specificity of membrane binding drastically differs. However, in both these proteins the PH domains work to modulate the activity of their catalytic core upon interaction with either phosphoinositol lipids or G protein activators. These observations show that these PH domains are not simply binding sites tethered onto their host enzyme but are intimately associated with their catalytic core. This property may be true for other PH domains.     

Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation

Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin α(IIb)β(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation.     

Rescue of the aggregation prone Itk Pleckstrin Homology domain by two mutations derived from the related kinases, Btk and Tec

IL-2 inducible T-cell kinase (Itk) is a Tec family non-receptor tyrosine kinase involved in signaling downstream of the T-cell receptor. Itk contains an amino-terminal Pleckstrin Homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate, recruiting Itk to the plasma membrane upon T-cell receptor activation. In addition to phosphoinositide binding, accumulating data suggest that the Itk PH domain likely mediates additional interactions outside of the phosphoinositide ligand binding pocket. The structural basis for additional PH domain functions remains elusive because of the poor recombinant expression and in vitro solution behavior of the Itk PH domain. Here, we determine that the lone α-helix in the Itk PH domain is responsible for the poor solution properties and that mutation of just two residues in the Itk α-helix to the corresponding amino acids in Btk or Tec dramatically improves the soluble recombinant expression and solution behavior of the Itk PH domain. We present this double mutant as a valuable tool to characterize the structure and function of the Itk PH domain. It is also interesting to note that the precise sites of mutation identified in this study appear as somatic mutations associated with cancerous tissue. Collectively, the findings suggest that the two helical residues in the Itk PH domain may serve an important and unique structural role in wild-type Itk that differentiates this tyrosine kinase from its related family members.     

Crystal Structure of the Pleckstrin Homology Domain from the Ceramide Transfer Protein: Implications for Conformational Change upon Ligand Binding

Ceramide transfer protein (CERT) is responsible for the nonvesicular trafficking of ceramide from the endoplasmic reticulum (ER) to the trans Golgi network where it is converted to sphingomyelin (SM). The N-terminal pleckstrin homology (PH) domain is required for Golgi targeting of CERT by recognizing the phosphatidylinositol 4-phosphate (PtdIns(4)P) enriched in the Golgi membrane. We report a crystal structure of the CERT PH domain. This structure contains a sulfate that is hydrogen bonded with residues in the canonical ligand-binding pocket of PH domains. Our nuclear magnetic resonance (NMR) chemical shift perturbation (CSP) analyses show sulfate association with CERT PH protein resembles that of PtdIns(4)P, suggesting that the sulfate bound structure likely mimics the holo form of CERT PH protein. Comparison of the sulfate bound structure with the apo form solution structure shows structural rearrangements likely occur upon ligand binding, suggesting conformational flexibility in the ligand-binding pocket. This structural flexibility likely explains CERT PH domain’s low affinity for PtdIns(4)P, a property that is distinct from many other PH domains that bind to their phosphoinositide ligands tightly. This unique structural feature of CERT PH domain is probably tailored towards the transfer activity of CERT protein where it needs to shuttle between ER and Golgi and therefore requires short resident time on ER and Golgi membranes.     

A single circularly permuted GFP sensor for inositol-1,3,4,5-tetrakisphosphate based on a split PH domain

A fluorescent sensor for the detection of inositol-1,3,4,5-tetrakisphosphate, Ins(1,3,4,5)P₄, was constructed from a split PH domain and a single circularly permuted GFP. A structure-based design was conducted to transduce a ligand-induced subtle structural perturbation of the split PH domain to an alteration in the population of the protonated and the deprotonated states of the GFP chromophore. Excitation of each distinct absorption band corresponding to the protonated or the deprotonated state of GFP resulted an increase and a decrease, respectively, in the intensity of emission spectra upon addition of Ins(1,3,4,5)P₄ to the split PH domain-based sensor. The Ins(1,3,4,5)P₄ sensor retained the ligand affinity and the selectivity of the parent PH domain, and realized the ratiometric fluorescence detection of Ins(1,3,4,5)P₄.     

Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx and Hox proteins

Direct auto- and cross-regulatory interactions between Hox genes serve to establish and maintain segmentally restricted patterns in the developing hindbrain. Rhombomere r4-specific expression of both Hoxb1 and Hoxb2 depends upon bipartite cis Hox response elements for the group 1 paralogous proteins, Hoxal and Hoxbl. The DNA-binding ability and selectivity of these proteins depend upon the formation of specific heterodimeric complexes with members of the PBC homeodomain protein family (Pbx genes). The r4 enhancers from Hoxb1 and Hoxb2 have the same activity, but differ with respect to the number and organisation of bipartite Pbx/Hox (PH) sites required, suggesting the intervention of other components/sequences. We report here that another family of homeodomain proteins (TALE, Three-Amino acids-Loop-Extension: Prep1, Meis, HTH), capable of dimerizing with Pbx/EXD, is involved in the mechanisms of r4-restricted expression. We show that: (1) the r4-specific Hoxb1 and Hoxb2 enhancers are complex elements containing separate PH and Prep/Meis (PM) sites; (2) the PM site of the Hoxb2, but not Hoxb1, enhancer is essential in vivo for r4 expression and also influences other sites of expression; (3) both PM and PH sites are required for in vitro binding of Prepl-Pbx and formation and binding of a ternary Hoxbl-Pbxla (or 1b)-Prepl complex. (4) A similar ternary association forms in nuclear extracts from embryonal P19 cells, but only upon retinoic acid induction. This requires synthesis of Hoxbl and also contains Pbx with either Prepl or Meisl. Together these findings highlight the fact that PM sites are found in close proximity to bipartite PH motifs in several Hox responsive elements shown to be important in vivo and that such sites play an essential role in potentiating regulatory activity in combination with the PH motifs.     

Crystal structure of kindlin-2 PH domain reveals a conformational transition for its membrane anchoring and regulation of integrin activation

Kindlin-2 belongs to a subfamily of FERM domain containing proteins, which plays key roles in activating integrin transmembrane receptors and mediating cell adhesion. Compared to conventional FERM domains, kindlin-2 FERM contains an inserted pleckstrin homology (PH) domain that specifically binds to phosphatidylinositol (3,4,5) trisphosphate (PIP3) and regulates the kindlin-2 function. We have determined the crystal structure of kindlin-2 PH domain at 1.9 ? resolution, which reveals a conserved PH domain fold with a highly charged and open binding pocket for PIP3 head group. Structural comparison with a previously reported solution structure of kindlin-2 PH domain bound to PIP3 head group reveals that upon PIP3 insertion, there is a significant conformational change of both the highly positively charged loop at the entry of the PIP3 binding pocket and the entire β barrel of the PH domain. We propose that such “induced-fit” type change is crucial for the tight binding of PIP3 to anchor kindlin-2 onto the membrane surface, thereby promoting its binding to integrins. Our results provide important structural insight into kindlin-2-mediated membrane anchoring and integrin activation.     

Tissue Level Compartmentation of (R)-Amygdalin and Amygdalin Hydrolase Prevents Large-Scale Cyanogenesis in Undamaged Prunus Seeds

Plum (Prunus domestica) seeds, which contain the cyanogenic diglucoside (R)-amygdalin and lesser amounts of the corresponding monoglucoside (R)-prunasin, release the respiratory toxin HCN upon tissue disruption. Amygdalin hydrolase (AH) and prunasin hydrolase (PH), two specific [beta]-glucosidases responsible for hydrolysis of these glucosides, were purified to near homogeneity by concanavalin A-Sepharose 4B and carboxymethyl-cellulose chromatography. Both proteins appear as polypeptides with molecular masses of 60 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but they exhibit different isoelectric points (PH, 5.6-6.0; AH, 7.8-8.2). AH and PH were localized within mature plum seeds by tissue printing, histochemistry, and silver-enhanced immunogold labeling. As was previously shown in black cherry (Prunus serotina) seeds (E.Swain, C.P. Li, J.E. Poulton [1992] Plant Physiol 100: 291-300), AH and PH are restricted to protein bodies of specific procambial cells and are absent from the cotyledonary parenchyma, bundle sheath, and endosperm cells. In contrast, the cyanogenic glycosides in both plum and black cherry seeds, which were detected by tissue printing, occur solely in the cotyledonary parenchyma and are absent from the procambium and endosperm. It is concluded that tissue level compartmentation prevents large-scale cyanoglycoside hydrolysis in intact Prunus seeds.     

RACK1, a protein kinase C scaffolding protein, interacts with the PH domain of p120GAP

The Ras GTPase-activating protein p120GAP is a multidomain protein consisting of a variety of noncatalytic domains that may be involved in its regulation. RACK1 is a membrane-associated protein that binds the C2 domain of PKC and is related in sequence to the beta subunit of heterotrimeric G-proteins which has been implicated in binding to PH domains. Because p120GAP contains both PH and C2/CaLB domains we determined whether it is also a RACK1 binding protein. Coimmunoprecipitation experiments indicate that p120GAP associates with RACK1, whereas PH or C2/CaLB domain deletion mutants do not. A fusion protein containing the GAP PH domain bound to endogenous RACK1 in lysates in a concentration-dependent manner and directly associated with recombinant RACK1. Finally, serine/threonine phosphorylation appears to be involved in regulating this association. These results suggest that p120GAP and RACK1 interact in vivo in a manner dependent upon both the PH and C2/CaLB domains of GAP.     

Structural Insights into the Activation of the RhoA GTPase by the Lymphoid Blast Crisis (Lbc) Oncoprotein

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and ¹H,¹³C,¹⁵N-based NMR spectroscopy. The two domains assume a “chair” shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.     

Point mutations in the split PLC-gamma1 PH domain modulate phosphoinositide binding

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal (PH(1)) and a split PH domain (nPH(2) and cPH(2)). We previously reported that the split PH domain of PLC-gamma1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P(2), we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-gamma1 nPH(2) domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-gamma1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-gamma1 molecules showed reduced PI(4,5)P(2) hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH(1) and nPH(2) domains are responsible for membrane-targeted translocation of PLC-gamma1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro(500) and His(503) are critical for binding of PLC-gamma1 to one of its substrates, PI(4,5)P(2) in the membrane.     

Novel Inhibitors Induce Large Conformational Changes of GAB1 Pleckstrin Homology Domain and Kill Breast Cancer Cells

The Grb2-associated binding protein 1 (GAB1) integrates signals from different signaling pathways and is over-expressed in many cancers, therefore representing a new therapeutic target. In the present study, we aim to target the pleckstrin homology (PH) domain of GAB1 for cancer treatment. Using homology models we derived, high-throughput virtual screening of five million compounds resulted in five hits which exhibited strong binding affinities to GAB1 PH domain. Our prediction of ligand binding affinities is also in agreement with the experimental K _D values. Furthermore, molecular dynamics studies showed that GAB1 PH domain underwent large conformational changes upon ligand binding. Moreover, these hits inhibited the phosphorylation of GAB1 and demonstrated potent, tumor-specific cytotoxicity against MDA-MB-231 and T47D breast cancer cell lines. This effort represents the discovery of first-in-class GAB1 PH domain inhibitors with potential for targeted breast cancer therapy and provides novel insights into structure-based approaches to targeting this protein.     

Primary hyperoxaluria type 1: AGT mistargeting highlights the fundamental differences between the peroxisomal and mitochondrial protein import pathways

Primary hyperoxaluria type 1 (PH1) is an atypical peroxisomal disorder, as befits a deficiency of alanine:glyoxylate aminotransferase (AGT), which is itself an atypical peroxisomal enzyme. PH1 is characterized by excessive synthesis and excretion of the metabolic end-product oxalate and the progressive accumulation of insoluble calcium oxalate in the kidney and urinary tract. Disease in many patients is caused by a unique protein trafficking defect in which AGT is mistargeted from peroxisomes to mitochondria, where it is metabolically ineffectual, despite remaining catalytically active. Although the peroxisomal import of human AGT is dependent upon the PTS1 import receptor PEX5p, its PTS1 is exquisitely specific for mammalian AGT, suggesting the presence of additional peroxisomal targeting information elsewhere in the AGT molecule. This and many other functional peculiarities of AGT are probably a consequence of its rather chequered evolutionary history, during which much of its time has been spent being a mitochondrial, rather than a peroxisomal, enzyme. Analysis of the molecular basis of AGT mistargeting in PH1 has thrown into sharp relief some of the fundamental differences between the requirements of the peroxisomal and mitochondrial protein import pathways, particularly the properties of peroxisomal and mitochondrial matrix targeting sequences and the different conformational limitations placed upon importable cargos.     

The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo

UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5)P(2) through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P(2) binding pocket of the PH domain, resulting in greatly reduced preferential binding to PI(4,5)P(2)in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P(2) levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PI(4,5)P(2) binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PI(4,5)P(2) levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PI(4,5)P(2) binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo.     

Expression of phosphatidylinositol (4,5) bisphosphate-specific pleckstrin homology domains alters direction but not the level of axonal transport of mitochondria

Axonal transport of membranous organelles such as mitochondria is essential for neuron viability and function. How signaling mechanisms regulate or influence mitochondrial distribution and transport is still largely unknown. We observed an increase in the distal distribution of mitochondria in neurons upon the expression of pleckstrin homology (PH) domains of phospholipase Cdelta1 (PLCdelta-PH) and spectrin (spectrin-PH). Quantitative analysis of mitochondrial transport showed that specific binding of PH domains to phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2) but not 3' phosphorylated phosphatidylinositol species enhanced plus-end-directed transport of mitochondria two- to threefold and at the same time decreased minus-end-directed transport of mitochondria along axonal microtubules (MTs) without altering the overall level of motility. Further, the velocity and duration of mitochondrial transport plus the association of molecular motors with mitochondria remained unchanged by the expression of PH domains. Thus, PtdIns(4,5)P2-specific PH domains caused an increase in distal mitochondria by disturbing the balance of plus- and minus-end-directed transport rather than directly affecting the molecular machinery involved. Taken together our data reveal that level and directionality of transport are separable and that PtdIns(4,5)P2 has a novel role in regulation of the directionality of axonal transport of mitochondria.     

In vivo functional analysis of the daughter of sevenless protein in receptor tyrosine kinase signaling

One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains a N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation suggested that Dos belongs to the family of multisite adaptor proteins that include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. Here, we studied the structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes by expressing mutated dos transgenes in the fly. We show that mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-γ (PLC-γ) and the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, we assayed whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a specific function of the PH domain in regulating signal transduction.     

Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients

The large GTPase dynamin has an important membrane scission function in receptor‐mediated endocytosis and other cellular processes. Self‐assembly on phosphoinositide‐containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin‐homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C‐terminal α‐helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either ‘sensitize’ dynamin to lipid stimulation or elevate basal GTPase rates by promoting self‐assembly and thus rendering dynamin no longer lipid responsive. We also describe a low‐resolution structure of dimeric dynamin from small‐angle X‐ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self‐assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.     

Novel role for RNase PH in the degradation of structured RNA

Escherichia coli contains multiple 3' to 5' RNases, of which two, RNase PH and polynucleotide phosphorylase (PNPase), use inorganic phosphate as a nucleophile to catalyze RNA cleavage. It is known that an absence of these two enzymes causes growth defects, but the basis for these defects has remained undefined. To further an understanding of the function of these enzymes, the degradation pattern of different cellular RNAs was analyzed. It was observed that an absence of both enzymes results in the appearance of novel mRNA degradation fragments. Such fragments were also observed in strains containing mutations in RNase R and PNPase, enzymes whose collective absence is known to cause an accumulation of structured RNA fragments. Additional experiments indicated that the growth defects of strains containing RNase R and PNPase mutations were exacerbated upon RNase PH removal. Taken together, these observations suggested that RNase PH could play a role in structured RNA degradation. Biochemical experiments with RNase PH demonstrated that this enzyme digests through RNA duplexes of moderate stability. In addition, mapping and sequence analysis of an mRNA degradation fragment that accumulates in the absence of the phosphorolytic enzymes revealed the presence of an extended stem-loop motif at the 3' end. Overall, these results indicate that RNase PH plays a novel role in the degradation of structured RNAs and provides a potential explanation for the growth defects caused by an absence of the phosphorolytic RNases.