A Comprehensive Prognostic and Immune Infiltration Analysis of RBM4 in Pan-Cancer.

BACKGROUND: Aberrant splicing has been closely associated with human cancer, though the precise underlying mechanisms linking the two remain not fully understood. Investigating the role of splicing factors in cancer progression may aid in the development of targeted therapies for dysregulated splicing, thereby opening up new avenues for cancer treatment. RNA-binding motif 4 (RBM4) has been identified as a critical participant in the condensin II complex, which is involved in chromosome condensation and stabilization during mitosis. Its significance in tumors is currently gaining attention. The genetic characteristics of RBM4 suggest its potential to elucidate the malignant progression of tumors in a broader context, encompassing various types of cancer, known as pan-cancer. METHODS: This study aims to comprehensively explore the potential function of RBM4 in pan-cancer by leveraging existing databases such as The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). RESULTS: RBM4 is found to be overexpressed in almost all tumors and exhibits significant prognostic and diagnostic efficacy. The correlation between RBM4 and immune signatures, including immune cell infiltration and immune checkpoint genes, indicates that RBM4 could serve as a guiding factor for immunotherapy. CONCLUSIONS: As a member of the pan-oncogene, RBM4 has the potential to become a biomarker and therapeutic target for various malignant tumors, offering novel possibilities for precision medicine.

RNA binding motif 4 inhibits the replication of ebolavirus by directly targeting 3'-leader region of genomic RNA.

Ebola virus (EBOV) belongs to Filoviridae family possessing single-stranded negative-sense RNA genome, which is a serious threat to human health. Nowadays, no therapeutics have been proven to be successful in efficiently decreasing the mortality rate. RNA binding proteins (RBPs) are reported to participate in maintaining cell integrity and regulation of viral replication. However, little is known about whether and how RBPs participate in regulating the life cycle of EBOV. In our study, we found that RNA binding motif protein 4 (RBM4) inhibited the replication of EBOV in HEK293T and Huh-7 cells by suppressing viral mRNA production. Such inhibition resulted from the direct interaction between the RRM1 domain of RBM4 and the "CU" enrichment elements located in the PE1 and TSS of the 3'-leader region within the viral genome. Simultaneously, RBM4 could upregulate the expression of some cytokines involved in the host innate immune responses to synergistically exert its antiviral function. The findings therefore suggest that RBM4 might serve as a novel target of anti-EBOV strategy.

Structural and Dynamical Basis of VP35-RBD Inhibition by Marine Fungi Compounds to Combat Marburg Virus Infection.

The Marburg virus (MBV), a deadly pathogen, poses a serious threat to world health due to the lack of effective treatments, calling for an immediate search for targeted and efficient treatments. In this study, we focused on compounds originating from marine fungi in order to identify possible inhibitory compounds against the Marburg virus (MBV) VP35-RNA binding domain (VP35-RBD) using a computational approach. We started with a virtual screening procedure using the Lipinski filter as a guide. Based on their docking scores, 42 potential candidates were found. Four of these compounds-CMNPD17596, CMNPD22144, CMNPD25994, and CMNPD17598-as well as myricetin, the control compound, were chosen for re-docking analysis. Re-docking revealed that these particular compounds had a higher affinity for MBV VP35-RBD in comparison to the control. Analyzing the chemical interactions revealed unique binding properties for every compound, identified by a range of Pi-cation interactions and hydrogen bond types. We were able to learn more about the dynamic behaviors and stability of the protein-ligand complexes through a 200-nanosecond molecular dynamics simulation, as demonstrated by the compounds' consistent RMSD and RMSF values. The multidimensional nature of the data was clarified by the application of principal component analysis, which suggested stable conformations in the complexes with little modification. Further insight into the energy profiles and stability states of these complexes was also obtained by an examination of the free energy landscape. Our findings underscore the effectiveness of computational strategies in identifying and analyzing potential inhibitors for MBV VP35-RBD, offering promising paths for further experimental investigations and possible therapeutic development against the MBV.

Theileria annulata subtelomere-encoded variable secreted protein-TA05560 interacts with bovine RNA binding motif protein 39 (RBM39).

Theileria annulata is the only eukaryotic pathogen able to transform bovine leukocytes, including B cells, macrophages and dendritic cells. T. annulata-transformed cells exhibit several cancer-like phenotypes, such as hyperproliferation, immortalization and dissemination. Although several parasite factors involved in bovine cell transformation have been explored, the roles of subtelomere-encoded variable secreted proteins (SVSPs) of the parasite in host-cell interactions are largely unknown. In the present study, the target molecule TA05560, a member of the SVSP multigene family of T. annulata, was identified at the mRNA level during different life cycles through a quantitative real-time PCR assay, and the subcellular distribution of TA05560 was examined via confocal microscopy. The results showed that the parasite molecule TA05560 was transcribed mainly in the schizont stage of T. annulata infection, and the protein was distributed in the nucleus and cytoplasm of the parasitized cells. The potential host cell proteins that interact with TA05560 were screened using the yeast two-hybrid system, and the direct interaction between TA05560 and its prey protein, Bos taurus RNA binding motif protein 39 (RBM39) was further identified in HEK293T cells by using confocal microscopy, coimmunoprecipitation and bimolecular fluorescence complementation assays. Moreover, the interaction between TA05560 and its host protein was observed in T. annulata-infected cells via confocal microscopy. Therefore, our study is the first to show that the T. annulata-secreted TA05560 protein directly binds to both the exogenous and endogenous host cell molecule RBM39, laying the foundation for exploring host-parasite interactions and understanding the transformation mechanisms induced by T. annulata and other transforming parasites.

The roles of FUS-RNA binding domain and low complexity domain in RNA-dependent phase separation.

Fused in sarcoma (FUS) is an archetypal phase separating protein asymmetrically divided into a low complexity domain (LCD) and an RNA binding domain (RBD). Here, we explore how the two domains contribute to RNA-dependent phase separation, RNA recognition, and multivalent complex formation. We find that RBD drives RNA-dependent phase separation but forms large and irregularly shaped droplets that are rescued by LCD in trans. Electrophoretic mobility shift assay (EMSA) and single-molecule fluorescence assays reveal that, while both LCD and RBD bind RNA, RBD drives RNA engagement and multivalent complex formation. While RBD alone exhibits delayed RNA recognition and a less dynamic RNP complex compared to full-length FUS, LCD in trans rescues full-length FUS activity. Likewise, cell-based data show RBD forms nucleolar condensates while LCD in trans rescues the diffuse nucleoplasm localization of full-length FUS. Our results point to a regulatory role of LCD in tuning the RNP interaction and buffering phase separation.

The RNA-binding motif protein 14 regulates telomere integrity at the interface of TERRA and telomeric R-loops.

Telomeric repeat-containing RNA (TERRA) and its formation of RNA:DNA hybrids (or TERRA R-loops), influence telomere maintenance, particularly in human cancer cells that use homologous recombination-mediated alternative lengthening of telomeres. Here, we report that the RNA-binding motif protein 14 (RBM14) is associated with telomeres in human cancer cells. RBM14 negatively regulates TERRA expression. It also binds to TERRA and inhibits it from forming TERRA R-loops at telomeres. RBM14 depletion has several effects, including elevated TERRA levels, telomeric R-loops, telomere dysfunction-induced DNA damage foci formation, particularly in the presence of DNA replication stress, pRPA32 accumulation at telomeres and telomere signal-free ends. Thus, RBM14 protects telomere integrity via modulating TERRA levels and its R-loop formation at telomeres.

RNA-binding motif protein 10 inactivates c-Myc by partnering with ribosomal proteins uL18 and uL5.

RNA-binding motif protein 10 (RBM10) is a frequently mutated tumor suppressor in lung adenocarcinoma (LUAD). Yet, it remains unknown whether cancer-derived mutant RBM10 compromises its tumor suppression function and, if so, the molecular insight of the underlying mechanisms. Here, we show that wild-type RBM10 suppresses lung cancer cell growth and proliferation by inactivating c-Myc that is essential for cancer cell survival. RBM10 directly binds to c-Myc and promotes c-Myc's ubiquitin-dependent degradation, while RBM10 knockdown leads to the induction of c-Myc level and activity. This negative action on c-Myc is further boosted by ribosomal proteins (RPs) uL18 (RPL5) and uL5 (RPL11) via their direct binding to RBM10. Cancer-derived mutant RBM10-I316F fails to bind to uL18 and uL5 and to inactivate c-Myc, thus incapable of suppressing tumorigenesis. Our findings uncover RBM10 as a pivotal c-Myc repressor by cooperating with uL18 and uL5 in lung cancer cells, as its failure to do so upon mutation favors tumorigenesis.

Molecular basis of RNA-binding and autoregulation by the cancer-associated splicing factor RBM39.

Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy but requires high levels of the adaptor protein DCAF15. Consequently, novel approaches to deplete RBM39 in an DCAF15-independent manner are required. Here, we uncover that RBM39 autoregulates via the inclusion of a poison exon into its own pre-mRNA and identify the cis-acting elements that govern this regulation. We also determine the NMR solution structures of RBM39's tandem RNA recognition motifs (RRM1 and RRM2) bound to their respective RNA targets, revealing how RRM1 recognises RNA stem loops whereas RRM2 binds specifically to single-stranded N(G/U)NUUUG. Our results support a model where RRM2 selects the 3'-splice site of a poison exon and the RRM3 and RS domain stabilise the U2 snRNP at the branchpoint. Our work provides molecular insights into RBM39-dependent 3'-splice site selection and constitutes a solid basis to design alternative anti-cancer therapies.

SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b.

The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding-prevented PKR activation, inhibited ß-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera.

Affinity-enhanced RNA-binding domains as tools to understand RNA recognition.

Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to use conservative mutations that enhance the affinity of RNA-binding domains to overcome this limitation. As a proof of principle, we have designed and validated an affinity-enhanced K-homology (KH) domain mutant of the fragile X syndrome protein FMRP, a key regulator of neuronal development, and used this mutant to determine the domain's sequence preference and to explain FMRP recognition of specific RNA motifs in the cell. Our results validate our concept and our nuclear magnetic resonance (NMR)-based workflow. While effective mutant design requires an understanding of the underlying principles of RNA recognition by the relevant domain type, we expect the method will be used effectively in many RNA-binding domains.

RNA-binding motif protein 24 inhibits HBV replication in vivo.

Despite the extensive use of effective vaccines and antiviral drugs, chronic hepatitis B virus (HBV) infection continues to pose a serious threat to global public health. Therapies with novel mechanisms of action against HBV are being explored for achieving a functional cure. In this study, five murine models of HBV replication were used to investigate the inhibitory effect of RNA binding motif protein 24 (RBM24) on HBV replication. The findings revealed that RBM24 serves as a host restriction factor and suppresses HBV replication in vivo. The transient overexpression of RBM24 in hydrodynamics-based mouse models of HBV replication driven by the CMV or HBV promoters suppressed HBV replication. Additionally, the ectopic expression of RBM24 decreased viral accumulation and the levels of HBV covalently closed circular DNA (cccDNA) in an rcccDNA mouse model. The liver-directed transduction of adeno-associated viruses (AAV)-RBM24 mediated the stable hepatic expression of RBM24 in pAAV-HBV1.2 and HBV/tg mouse models, and markedly reduced the levels of HBV cccDNA and other viral indicators. Altogether, these findings revealed that RBM24 inhibits the replication of HBV in vivo, and RBM24 may be a potential therapeutic target for combating HBV infections.

RNA-binding domain 2 of nucleolin is important for the autophagy induction of curcumol in nasopharyngeal carcinoma cells.

BACKGROUND & AIMS: Excessive autophagy induces cell death and is regarded as the treatment of cancer therapy. We have confirmed that the anti-cancer mechanism of curcumol is related to autophagy induction. As the main target protein of curcumol, RNA binding protein nucleolin (NCL) interacted with many tumor promoters accelerating tumor progression. However, the role of NCL in cancer autophagy and in curcumol's anti-tumor effects haven't elucidated. The purpose of the study is to identify the role of NCL in nasopharyngeal carcinoma autophagy and reveal the immanent mechanisms of NCL played in cell autophagy. METHODS & RESULTS: In the current study, we have found that NCL was markedly upregulated in nasopharyngeal carcinoma (NPC) cells. NCL overexpression effectively attenuated the level of autophagy in NPC cells, and NCL silence or curcumol treatment obviously aggravated the autophagy of NPC cells. Moreover, the attenuation of NCL by curcumol lead a significant suppression on PI3K/AKT/mTOR signaling pathway in NPC cells. Mechanistically, NCL was found to be directly interact with AKT and accelerate AKT phosphorylation, which caused the activation of the PI3K/AKT/mTOR pathway. Meanwhile, the RNA Binding Domain (RBD) 2 of NCL interacts with Akt, which was also influenced by curcumol. Notably, the RBDs of NCL delivered AKT expression was related with cell autophagy in the NPC. CONCLUSION: The results demonstrated that NCL regulated cell autophagy was related with interaction of NCL and Akt in NPC cells. The expression of NCL play an important role in autophagy induction and further found that was associated with its effect on NCL RNA-binding domain 2. This study may provide a new perspective on the target protein studies for natural medicines and confirm the effect of curcumol not only regulating the expression of its target protein, but also influencing the function domain of its target protein.

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy.

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knockin (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We showed that Rbm20ΔRS mice manifested DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 was mis-localized to the sarcoplasm in Rbm20ΔRS mouse hearts and formed RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif showed similar mis-splicing of major RBM20 target genes but did not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrated that only DCM-associated mutations in the RS domain facilitated RBM20 nucleocytoplasmic transport and promoted granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain of RBM20. Mutation analysis of phosphorylation sites in the RS domain suggested that this modification may be dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

Study on the protective effect of RNA-binding motif protein 3 in mild hypothermia oxygen-glucose deprivation/reoxygenation cell model.

Mild hypothermia is proven neuroprotective in clinical practice. While hypothermia leads to the decrease of global protein synthesis rate, it upregulates a small subset of protein including RNA-binding motif protein 3 (RBM3). In this study, we treated mouse neuroblastoma cells (N2a) with mild hypothermia before oxygen-glucose deprivation/reoxygenation (OGD/R) and discovered the decrease of apoptosis rate, down-regulation of apoptosis-associated protein and enhancement of cell viability. Overexpression of RBM3 via plasmid exerted similar effect while silencing RBM3 by siRNAs partially reversed the protective effect exerted by mild hypothermia pretreatment. The protein level of Reticulon 3(RTN3), a downstream gene of RBM3, also increased after mild hypothermia pretreatment. Silencing RTN3 weakened the protective effect of mild hypothermia pretreatment or RBM3 overexpression. Also, the protein level of autophagy gene LC3B increased after OGD/R or RBM3 overexpression while silencing RTN3 decreased this trend. Furthermore, immunofluorescence observed enhanced fluorescence signal of LC3B and RTN3 as well as a large number of overlaps after RBM3 overexpressing. In conclusion, RBM3 plays a cellular protective role by regulating apoptosis and viability via its downstream gene RTN3 in the hypothermia OGD/R cell model and autophagy may participate in it.

RNA-binding proteins that lack canonical RNA-binding domains are rarely sequence-specific.

Thousands of RNA-binding proteins (RBPs) crosslink to cellular mRNA. Among these are numerous unconventional RBPs (ucRBPs)-proteins that associate with RNA but lack known RNA-binding domains (RBDs). The vast majority of ucRBPs have uncharacterized RNA-binding specificities. We analyzed 492 human ucRBPs for intrinsic RNA-binding in vitro and identified 23 that bind specific RNA sequences. Most (17/23), including 8 ribosomal proteins, were previously associated with RNA-related function. We identified the RBDs responsible for sequence-specific RNA-binding for several of these 23 ucRBPs and surveyed whether corresponding domains from homologous proteins also display RNA sequence specificity. CCHC-zf domains from seven human proteins recognized specific RNA motifs, indicating that this is a major class of RBD. For Nudix, HABP4, TPR, RanBP2-zf, and L7Ae domains, however, only isolated members or closely related homologs yielded motifs, consistent with RNA-binding as a derived function. The lack of sequence specificity for most ucRBPs is striking, and we suggest that many may function analogously to chromatin factors, which often crosslink efficiently to cellular DNA, presumably via indirect recruitment. Finally, we show that ucRBPs tend to be highly abundant proteins and suggest their identification in RNA interactome capture studies could also result from weak nonspecific interactions with RNA.

RNA-binding motif 4 promotes angiogenesis in HCC by selectively activating VEGF-A expression.

Increased angiogenesis in the liver plays a critical role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanism underlying increased angiogenesis in HCC is not well understood. Current study was designed to identify the potential angiogenic effect of RNA-binding motif 4 (RBM4)through a small-scale overexpression screening, followed by comparison of the expression level of RBM4 in cancer and adjacent tissues in multiple malignancies to explore the relationship between RBM4 and CD31 protein expression level and related clinical indicators, and understand the role of RBM4 in the hepatocellular carcinoma. To understand the specific mechanism of RBM4 in detail, transcriptome sequencing, mass spectrometry and multiple molecular cytological studies were performed. These cellular level results were verified by experiments in animal models of nude mice. The increased expression of RBM4 in cancer tissues, suggested its use as a prognostic biomarker. The RBM4 expression was found to be strongly correlated with tumor microvessel density. Mechanistically, RBM4 mediated its effects via interaction with HNRNP-M through the latter's WDR15 domain, which then stabilized RelA/p65 mRNA. Consequently, RBM4 induced the activation of the NF-kB signaling pathway, upregulating the expression of proangiogenic factor VEGF-A. The results confirmed the mechanism by which RBM4 promotes angiogenesis in hepatocellular carcinoma suggesting RBM4 as a crucial promoter of angiogenesis in HCC, helping understand regulation of NF-kB signaling in HCC.

Cold-Induced RNA-Binding Protein and RNA-Binding Motif Protein 3: Two RNA Molecular Chaperones Closely Related to Reproductive Development and Reproductive System Diseases.

Cold-induced RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) have recently been reported to be involved in cold stress in mammals. These proteins are expressed at low levels in various normal cells, tissues, and organs but can be upregulated upon stimulation by multiple stressors. Studies have shown that CIRP and RBM3 are multifunctional RNA molecular chaperones with different biological functions in various physiological and pathophysiological processes, such as reproductive development, the inflammatory response, the immune response, nerve injury regulation, and tumorigenesis. This paper reviews recent studies on the structure, localization and correlation of CIRP and RBM3 with reproductive development and reproductive system diseases.

C-terminal determinants for RNA binding motif 7 protein stability and RNA recognition.

The 7SK ribonucleoprotein (RNP) is a critical regulator of eukaryotic transcription. Recently, RNA binding motif 7 (RBM7) containing an RNA recognition motif (RRM) was reported to associate with 7SK RNA and core 7SK RNP protein components in response to DNA damage. However, little is known about the mode of RBM7-7SK RNA recognition. Here, we found that RRM constructs containing extended C-termini have increased solubility compared to a minimal RRM construct, although these constructs aggregate in a temperature and concentration-dependent manner. Using solution NMR dynamics experiments, we identified additional structural features observed previously in crystal but not in solution structures. To identify potential RBM7-7SK RNA binding sites, we analyzed deposited data from in cellulo crosslinking experiments and found that RBM7 primarily crosslinks to the distal region of 7SK stem-loop 3 (SL3). Electrophoretic mobility shift assays and NMR chemical shift perturbation experiments showed weak binding to 7SK SL3 constructs in vitro. Together, these results provide new insights into RBM7 RRM folding and recognition of 7SK RNA.

11S Proteasome Activator REGγ Promotes Aortic Dissection by Inhibiting RBM3 (RNA Binding Motif Protein 3) Pathway.

BACKGROUND: Aortic dissection (AD) is a life-threatening cardiovascular disorder with high mortality and lacking underlying mechanisms or effective treatments. REGγ, the 11S proteasome activator known to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner, emerges as a new regulator in the cardiovascular system. METHODS: Using ß-aminopropionitrile (BAPN)-subjected REGγ knockout AD mice and Ang II (angiotensin II)-treated REGγ deficiency vascular smooth muscle cells (VSMCs) to explore the effect of REGγ in AD progression. RESULTS: REGγ was upregulated in mouse aorta of ß-aminopropionitrile-induced AD model in vivo and Ang II-treated VSMCs in vitro. REGγ deficiency ameliorated AD progression in ß-aminopropionitrile-induced mice by protecting against the switch in VSMCs from contractile to synthetic phenotype through suppressing RBM3 (RNA-binding motif protein 3) decay. Mechanically, REGγ interacted with and degraded the RNA-binding protein RBM3 directly, leading to decreased mRNA stability, lowered expression and transcriptional activity of transcription factor SRF (serum response factor), subsequently reduced transcription of VSMCs-specific contractile genes, α-SMA (alpha-smooth muscle actin) and SM22α (smooth muscle 22 alpha), caused the switch in VSMCs from contractile to synthetic phenotype and associated AD progression. Ablation of endogenous SRF or RBM3, or overexpressing exogenous RBM3 in VSMCs significantly blocked or reestablished the REGγ-dependent action on VSMCs phenotypic switch of Ang II stimulation in vitro. Furthermore, exogenously introducing RBM3 improved the switch in VSMCs from contractile to synthetic phenotype and associated AD features caused by REGγ in vivo. CONCLUSIONS: Our results demonstrated that REGγ promoted the switch in VSMCs from contractile to synthetic phenotype and AD progression by inhibiting RBM3-SRF pathway, indicated that modulating REGγ-proteasome activity may be a potential therapeutic approach for AD-associated cardiovascular dysfunction.

Target recognition by RNase E RNA-binding domain AR2 drives sRNA decay in the absence of PNPase.

The C-terminal domain (CTD) of the major endoribonuclease RNase E not only serves as a scaffold for the central RNA decay machinery in gram-negative bacteria but also mediates coupled degradation of small regulatory RNAs (sRNAs) and their cognate target transcripts following RNA chaperone Hfq-facilitated sRNA-mRNA base pairing. Despite the crucial role of RNase E CTD in sRNA-dependent gene regulation, the contribution of particular residues within this domain in recruiting sRNAs and mRNAs upon base pairing remains unknown. We have previously shown that in Escherichia coli, the highly conserved 3'-5'-exoribonuclease polynucleotide phosphorylase (PNPase) paradoxically stabilizes sRNAs by limiting access of RNase E to Hfq-bound sRNAs and by degrading target mRNA fragments that would otherwise promote sRNA decay. Here, we report that in the absence of PNPase, the RNA-binding region AR2 in the CTD is required for RNase E to initiate degradation of the Hfq-dependent sRNAs CyaR and RyhB. Additionally, we show that introducing mutations in either hfq that disrupts target mRNA binding to Hfq or the AR2 coding region of rne impairs RNase E binding to sRNAs. Altogether, our data support a model where sRNAs are recruited via bound mRNA targets to RNase E by its AR2 domain after Hfq catalyzes sRNA-mRNA pairing. These results also support our conclusion that in a PNPase-deficient strain, more rapid decay of sRNAs occurs due to accelerated pairing with mRNA targets as a consequence of their accumulation. Our findings provide insights into the mechanisms by which sRNAs and mRNAs are regulated by RNase E.