Blocking LBH expression causes replication stress and sensitizes triple-negative breast cancer cells to ATR inhibitor treatment.

Triple-negative (ER-PR-HER2-) breast cancers (TNBC) are highly aggressive and difficult to treat. TNBC exhibit high genomic instability, which enables them to adapt and become resistant to chemo/radiation therapy, leading to rapid disease relapse and mortality. The pro-survival factors that safeguard genome integrity in TNBC cells are poorly understood. LBH is an essential mammary stem cell-specific transcription regulator in the WNT pathway that is aberrantly overexpressed in TNBC, correlating with poor prognosis. Herein, we demonstrate a novel role for LBH in promoting TNBC cell survival. Depletion of LBH in multiple TNBC cell models triggered apoptotic cell death both in vitro and in vivo and led to S-G2M cell cycle delays. Mechanistically, LBH loss causes replication stress due to DNA replication fork stalling, leading to ssDNA breaks, ɣH2AX and 53BP1 nuclear foci formation, and activation of the ATR/CHK1 DNA damage response. Notably, ATR inhibition in combination with LBH downmodulation had a synergistic effect, boosting TNBC cell killing and blocking in vivo tumor growth. Our findings demonstrate, for the first time, that LBH protects the genome integrity of cancer cells by preventing replicative stress. Importantly, they uncover new synthetic lethal vulnerabilities in TNBC that could be exploited for future multi-modal precision medicine.

Suppression of multiple phenotypic and RNA processing defects by overexpression of oligoribonuclease in Escherichia coli.

Oligoribonuclease (Orn) is an essential ribonuclease (RNase) from Escherichia coli (E. coli), which plays a critical role in the conversion of short RNA molecules (NanoRNAs) to mononucleotides. Although no additional functions have been ascribed to Orn since its discovery nearly 50 years ago, it was observed in this study that the growth defects caused by a lack of two other RNases that do not digest NanoRNAs, polynucleotide phosphorylase, and RNase PH, could be suppressed by overexpression of Orn. Further analyses showed that overexpression of Orn can alleviate the growth defects caused by an absence of other RNases even when its expression was increased by a small degree, and it can carry out molecular reactions that are normally performed by RNase T and RNase PH. In addition, biochemical assays revealed that Orn can fully digest single-stranded RNAs within a variety of structural contexts. These studies provide new insights into Orn function and its ability to participate in multiple aspects of E. coli RNA metabolism.

Expression of the DeaD RNA Helicase Is Regulated at Multiple Levels through Its Long mRNA 5' Untranslated Region.

DEAD-box proteins (DBPs) are a prominent class of RNA remodeling proteins that alter RNA structure, a process they typically perform through an ATP-dependent RNA helicase activity. Although many DBPs have been characterized at the structural and functional level in detail, much less is known about how they are regulated. We previously showed that the mRNA for the Escherichia coli DeaD DBP contains an unusually long 5' untranslated region (5' UTR) of 838 nucleotides (nt) and that it is the primary RNA determinant of DeaD autoregulation. We speculated that such a long and complex 5' UTR might regulate deaD expression in additional ways. Here, we show that the deaD mRNA 5' UTR regulates deaD expression at two additional levels, temperature-dependent expression and through a stem-loop structure overlapping the start codon. These results support the hypothesis that a long 5' UTR can regulate gene expression through multiple mechanisms. IMPORTANCE The expression of genes is frequently regulated by determinants in the 5' UTR. Although many different regulatory mechanisms that operate via the 5' UTR have been described, the functional relevance of genes with long UTRs is less clear. Here, we show that the 838-nt-long 5' UTR in the deaD mRNA regulates the expression of DeaD at multiple levels. We propose that long UTRs originate to provide precise control of gene expression through multiple regulatory mechanisms, and they are indicators of the importance of their associated gene products for cellular adaptation to different environments.

Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination.

DEAD-box proteins (DBPs) are RNA remodeling factors associated with RNA helicase activity that are found in nearly all organisms. Despite extensive studies on the mechanisms used by DBPs to regulate RNA function, very little is known about how DBPs themselves are regulated. In this work, we have analyzed the expression and regulation of DeaD/CsdA, the largest of the DBPs in Escherichia coli (E. coli). We show that deaD transcription initiates 838 nt upstream of the start of the coding region. We have also found that DeaD is autoregulated through a negative feedback mechanism that operates both at the level of deaD mRNA stability and Rho-dependent transcription termination, and this regulation is dependent upon its mRNA 5' untranslated region (5' UTR). These findings suggest that DeaD might be regulating the conformation of its own mRNA through its RNA helicase activity to facilitate ribonuclease and Rho access to its 5' UTR.

RNase AM, a 5' to 3' exonuclease, matures the 5' end of all three ribosomal RNAs in E. coli.

Bacterial ribosomal RNAs (rRNAs) are transcribed as precursors and require processing by Ribonucleases (RNases) to generate mature and functional rRNAs. Although the initial steps of rRNA processing in Escherichia coli (E. coli) were described several decades ago, the enzymes responsible for the final steps of 5S and 23S rRNA 5'-end maturation have remained unknown. Here, I show that RNase AM, a recently identified 5' to 3' exonuclease, performs the last step of 5S rRNA 5'-end maturation. RNase AM was also found to generate the mature 5' end of 23S rRNA, subsequent to a newly identified prior processing step. Additionally, RNase AM was found to mature the 5' end of 16S rRNA, a reaction previously attributed to RNase G. These findings indicate a major role for RNase AM in cellular RNA metabolism and establish a biological role for the first 5' to 3' RNA exonuclease identified in E. coli.

A DEAD-box protein regulates ribosome assembly through control of ribosomal protein synthesis.

DEAD-box proteins (DBPs) comprise a large family of proteins that most commonly have been identified as regulators of ribosome assembly. The Escherichia coli DBP, SrmB, represents a model bacterial DBP whose absence impairs formation of the large ribosomal subunit (LSU). To define the basis for SrmB function, suppressors of the ribosomal defect of ΔsrmB strains were isolated. The major class of suppressors was found to map to the 5' untranslated region (UTR) of the rplM-rpsI operon, which encodes the ribosomal proteins (r-proteins) L13 and S9. An analysis of protein abundance indicated that both r-proteins are under-produced in the ΔsrmB strain, but are increased in these suppressors, implicating r-protein underproduction as the molecular basis for the observed ribosomal defects. Reduced r-protein synthesis was determined to be caused by intrinsic transcription termination within the rplM 5' UTR that is abrogated by SrmB. These results reveal a specific mechanism for DBP regulation of ribosomal assembly, indirectly mediated through its effects on r-protein expression.

Role of ribosome assembly in Escherichia coli ribosomal RNA degradation.

DEAD-Box proteins (DBPs) constitute a prominent class of RNA remodeling factors that play a role in virtually all aspects of RNA metabolism. To better define their cellular functions, deletions in the genes encoding each of the Escherichia coli DBPs were combined with mutations in genes encoding different Ribonucleases (RNases). Significantly, double-deletion strains lacking Ribonuclease R (RNase R) and either the DeaD or SrmB DBP were found to display growth defects and an enhanced accumulation of ribosomal RNA (rRNA) fragments. As RNase R is known to play a key role in removing rRNA degradation products, these observations initially suggested that these two DBPs could be directly involved in the same process. However, additional investigations indicated that DeaD and SrmB-dependent rRNA breakdown is caused by delays in ribosome assembly that increase the exposure of nascent RNAs to endonucleolytic cleavage. Consistent with this notion, mutations in factors known to be important for ribosome assembly also resulted in enhanced rRNA breakdown. Additionally, significant levels of rRNA breakdown products could be visualized in growing cells even in the absence of assembly defects. These findings reveal a hitherto unappreciated mechanism of rRNA degradation under conditions of both normal and abnormal ribosome assembly.

Otopathogenic Pseudomonas aeruginosa Enters and Survives Inside Macrophages.

Otitis media (OM) is a broad term describing a group of infectious and inflammatory disorders of the middle ear. Despite antibiotic therapy, acute OM can progress to chronic suppurative otitis media (CSOM) characterized by ear drum perforation and purulent discharge. Pseudomonas aeruginosa is the most common pathogen associated with CSOM. Although, macrophages play an important role in innate immune responses but their role in the pathogenesis of P. aeruginosa-induced CSOM is not known. The objective of this study is to examine the interaction of P. aeruginosa with primary macrophages. We observed that P. aeruginosa enters and multiplies inside human and mouse primary macrophages. This bacterial entry in macrophages requires both microtubule and actin dependent processes. Transmission electron microscopy demonstrated that P. aeruginosa was present in membrane bound vesicles inside macrophages. Interestingly, deletion of oprF expression in P. aeruginosa abrogates its ability to survive inside macrophages. Our results suggest that otopathogenic P. aeruginosa entry and survival inside macrophages is OprF-dependent. The survival of bacteria inside macrophages will lead to evasion of killing and this lack of pathogen clearance by phagocytes contributes to the persistence of infection in CSOM. Understanding host-pathogen interaction will provide novel avenues to design effective treatment modalities against OM.

Gateway role for rRNA precursors in ribosome assembly.

In Escherichia coli, rRNAs are initially transcribed with precursor sequences, which are subsequently removed through processing reactions. To investigate the role of precursor sequences, we analyzed ribosome assembly in strains containing mutations in the processing RNases. We observed that defects in 23S rRNA processing resulted in an accumulation of ribosomal subunits and caused a significant delay in ribosome assembly. These observations suggest that precursor residues in 23S rRNA control ribosome assembly and could be serving a regulatory role to couple ribosome assembly to rRNA processing. The possible mechanisms through which rRNA processing and ribosome assembly could be linked are discussed.

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.

Role of precursor sequences in the ordered maturation of E. coli 23S ribosomal RNA.

The maturation of ribosomal RNAs (rRNAs) is an important but incompletely understood process required for rRNAs to become functional. In order to determine the enzymes responsible for initiating 3' end maturation of 23S rRNA in Escherichia coli, we analyzed a number of strains lacking different combinations of 3' to 5' exo-RNases. Through these analyses, we identified RNase PH as a key effector of 3' end maturation. Further analysis of the processing reaction revealed that the 23S rRNA precursor contains a CC dinucleotide sequence that prevents maturation from being performed by RNase T instead. Mutation of this dinucleotide resulted in a growth defect, suggesting a strategic significance for this RNase T stalling sequence to prevent premature processing by RNase T. To further explore the roles of RNase PH and RNase T in RNA processing, we identified a subset of transfer RNAs (tRNAs) that contain an RNase T stall sequence, and showed that RNase PH activity is particularly important to process these tRNAs. Overall, the results obtained point to a key role of RNase PH in 23S rRNA processing and to an interplay between this enzyme and RNase T in the processing of different species of RNA molecules in the cell.

Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms.

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.

The outer membrane protein A (OmpA) of Yersinia pestis promotes intracellular survival and virulence in mice.

The plague bacterium Yersinia pestis has a number of well-described strategies to protect itself from both host cells and soluble factors. In an effort to identify additional anti-host factors, we employed a transposon site hybridization (TraSH)-based approach to screen 10(5)Y. pestis mutants in an in vitro infection system. In addition to loci encoding various components of the well-characterized type III secretion system (T3SS), our screen unambiguously identified ompA as a pro-survival gene. We go on to show that an engineered Y. pestis ΔompA strain, as well as a ΔompA strain of the closely related pathogen Yersinia pseudotuberculosis, have fully functioning T3SSs but are specifically defective in surviving within macrophages. Additionally, the Y. pestis ΔompA strain was out competed by the wild-type strain in a mouse co-infection assay. Unlike in other bacterial pathogens in which OmpA can promote adherence, invasion, or serum resistance, the OmpA of Y. pestis is restricted to enhancing intracellular survival. Our data show that OmpA of the pathogenic Yersinia is a virulence factor on par with the T3SS.

DEAD-box proteins from Escherichia coli exhibit multiple ATP-independent activities.

DEAD-box proteins (DBPs) are a widespread class of ATP-dependent RNA helicases that play a key role in unwinding RNA duplexes. In recent years, certain DBPs have also been found to exhibit activities that do not require ATP. To gain a better understanding of prokaryotic RNA metabolism, we investigated whether Escherichia coli DBPs harbor any ATP-independent activities. We show that each of the four E. coli DBPs tested in this study can accelerate the association of cRNA molecules, can stimulate strand displacement, and can function as an RNA chaperone without utilizing ATP. To the best of our knowledge, these prokaryotic DBPs constitute the first examples of proteins that harbor each of these three activities. The identification of these auxiliary functions indicates that the E. coli DBPs are versatile factors that possess significant RNA remodeling activity in addition to their canonical RNA helicase activity and might therefore participate in a greater variety of cellular processes than has been previously appreciated.

Functional and molecular analysis of Escherichia coli strains lacking multiple DEAD-box helicases.

DEAD-box RNA helicases are enzymes that unwind RNA duplexes and are found in virtually all organisms. Most organisms harbor multiple DEAD-box helicases, suggesting that these factors participate in distinct aspects of RNA metabolism. To define the individual and collective contribution of the five DEAD-box helicases in the bacterium Escherichia coli (E. coli), nonpolar deletion mutants lacking single or multiple DEAD-box genes were constructed. An analysis of the single-deletion strains indicated that the absence of either the DeaD or SrmB RNA helicase causes growth and/or ribosomal defects under typical laboratory growth conditions. The analysis of strains lacking multiple DEAD-box genes showed cumulative growth defects at low temperatures. A strain deleted for all five DEAD-box genes was also constructed for these studies, representing the first time all DEAD-box genes have been removed in any organism. Additional investigations revealed that the growth and ribosomal defects of such a DEAD-box deficient strain can be sharply attenuated under alternative conditions, indicating that the defects caused by a lack of DEAD-box genes are modulated by growth context.

Coordinated regulation of 23S rRNA maturation in Escherichia coli.

In Escherichia coli, rRNAs are transcribed as precursors and require processing at the 3' and 5' ends to generate mature RNA molecules. The largest of these RNAs, 23S rRNA, is matured at the 3' end by a set of exonucleases and at the 5' end by an unknown RNase. Whether the 3' and 5' maturation steps occur independently or are coupled has previously been unclear. By assessing the levels of precursors accumulating at the 3' and 5' ends, we provide evidence that these processes may be linked. Thus, each of several conditions that led to precursor accumulation at one end also did so at the other end. We also observed that each end undergoes maturation at similar rates, suggesting that the two processes could be coupled. Finally, we provide evidence that processing at the 3' end facilitates 5'-end maturation. A model to explain the basis for the observed directionality of the reactions is proposed. This information will aid in the search for the enzyme responsible for final maturation of the 5' end of 23S rRNA.

Identification and characterization of growth suppressors of Escherichia coli strains lacking phosphorolytic ribonucleases.

RNases are involved in critical aspects of RNA metabolism in all organisms. Two classes of RNases that digest RNA from an end (exo-RNases) are known: RNases that use water as a nucleophile to catalyze RNA degradation (hydrolytic RNases) and RNases that use inorganic phosphate (phosphorolytic RNases). It has been shown previously that the absence of the two known Escherichia coli phosphorolytic RNases, polynucleotide phosphorylase and RNase PH, leads to marked growth and ribosome assembly defects. To investigate the basis for these defects, a screen for growth suppressors was performed. The majority of suppressor mutations were found to lie within nsrR, which encodes a nitric oxide (NO)-sensitive transcriptional repressor. Further analysis showed that the suppressors function not by inactivating nsrR but by causing overexpression of a downstream gene that encodes a hydrolytic RNase, RNase R. Additional studies revealed that overexpression of another hydrolytic RNase, RNase II, similarly suppressed the growth defects. These results suggest that the requirement for phosphorolytic RNases for robust cellular growth and efficient ribosome assembly can be bypassed by increased expression of hydrolytic RNases.

Compensatory mutations in the L30e kink-turn RNA-protein complex.

The S. cerevisiae ribosomal protein L30e is an autoregulatory protein that binds to its own pre-mRNA and mature mRNA to inhibit splicing and translation, respectively. The L30e RNA-binding element is a stem-asymmetric loop-stem that forms a kink-turn. A bacterial genetic system was designed to test the ability of protein variants to repress the expression of reporter mRNAs containing the L30e RNA-binding element. Initial screens revealed that changes in several RNA nucleotides had a measurable effect on repression of the reporter by the wild type protein. RNA mutants that reduce repression were screened against libraries of randomly mutagenized L30e proteins. These screens identified a glycine to serine mutation of L30e, which specifically restores activity to an RNA variant containing a U that replaces a helix-capping G. Similarly, an asparagine to alanine mutation was found to suppress a substitution at a position where the L30e RNA nucleotide extends out into the protein pocket. In addition, a compensatory RNA mutation within a defective RNA variant was found. The identification of these suppressors provides new insights into the architecture of a functional binding element and its recognition by an important RNA-binding protein.

RNase E regulates the Yersinia type 3 secretion system.

Yersinia spp. use a type 3 secretion system (T3SS) to directly inject six proteins into macrophages, and any impairment of this process results in a profound reduction in virulence. We previously showed that the exoribonuclease polynucleotide phosphorylase (PNPase) was required for optimal T3SS functioning in Yersinia pseudotuberculosis and Yersinia pestis. Here we report that Y. pseudotuberculosis cells with reduced RNase E activity are likewise impaired in T3SS functioning and that phenotypically they resemble Delta pnp cells. RNase E does not affect expression levels of the T3SS substrates but instead, like PNPase, regulates a terminal event in the secretion pathway. This similarity, together with the fact that RNase E and PNPase can be readily copurified from Y. pseudotuberculosis cell extracts, suggests that these two RNases regulate T3SS activity through a common mechanism. This is the first report that RNase E activity impacts the T3SS as well as playing a more general role in infectivity.