Microneedle-Delivered PDA@Exo for Multifaceted Osteoarthritis Treatment via PI3K-Akt-mTOR Pathway.

Osteoarthritis (OA) is marked by cartilage deterioration, subchondral bone changes, and an inflammatory microenvironment. The study introduces the Microneedle-Delivered Polydopamine-Exosome (PDA@Exo MN), a therapeutic that not only preserves cartilage and promotes bone regeneration but also improves localized drug delivery through enhanced penetration capabilities. PDA@Exo MN shows strong reactive oxygen species (ROS) scavenging abilities and high biocompatibility, fostering osteogenesis and balancing anabolic and catabolic processes in cartilage. It directs macrophage polarization from M0 to the anti-inflammatory M2 phenotype. RNA sequencing of treated chondrocytes demonstrates restored cellular function and activated antioxidant responses, with modulated inflammatory pathways. The PI3K-AKT-mTOR pathway's activation, essential for PDA@Exo's effects, is confirmed via bioinformatics and Western blot. In vivo assessments robustly validate that PDA@Exo MN prevents cartilage degradation and OA progression, supported by histological assessments and micro-CT analysis, highlighting its disease-modifying impact. The excellent biocompatibility of PDA@Exo MN, verified through histological (H&E) and blood tests showing no organ damage, underscores its safety and efficacy for OA therapy, making it a novel and multifunctional nanomedical approach in orthopedics, characterized by organ-friendliness and biosecurity.

FOXC2-AS1/FOXC2 axis mediates matrix stiffness-induced trans-differentiation of hepatic stellate cells into fibrosis-promoting myofibroblasts.

Matrix stiffness is a central modulator of hepatic stellate cells (HSCs) activation and hepatic fibrogenesis. However, the long non-coding RNAs (lncRNAs)-regulated transcriptional factors linking matrix stiffness to alterations in HSCs phenotype are not completely understood. In this study, we investigated the effects of matrix stiffness on HSCs activation and its potential mechanism. Through analysis the RNA-seq data with human primary HSCs cultured on 0.4 kPa and 25.6 kPa hydrogel, we identified that forkhead box protein C2 (FOXC2) and its antisense lncRNA FXOC2-AS1 as the new mechanosensing transcriptional regulators that coordinate HSCs responses to the matrix stiffness, moreover, FOXC2 and FOXC2-AS1 expression were also elevated in human fibrosis and cirrhosis tissues. The matrix stiffness was sufficient to activate HSCs into myofibroblasts, resulting in nuclear accumulation of FOXC2. Disrupting FOXC2 and FOXC2-AS1 level abrogated stiffness-induced activation of HSCs. Further mechanistic studies displayed that stiffness-upregulated lncRNA FOXC2-AS1 had no influence on transcription of FOXC2. FOXC2-AS1 exerted its biological function through maintaining the RNA stability of FOXC2, and protecting FOXC2 mRNA from degradation by RNA exosome complex. Additionally, rescue assays confirmed that reintroduction of FOXC2 in FOXC2-AS1-depleted HSCs reversed the repression of FOXC2-AS1 knockdown on stiffness-induced HSCs activation. In AAV6-treated mice fibrotic models, targeting FOXC2 in vivo lead to a reduced degree of liver fibrosis. In sum, our study uncovers a reciprocal crosstalk between matrix stiffness and FOXC2-AS1/FOXC2 axis leading to modulation of HSCs mechanoactivation and liver fibrosis, and present AAV6 shRNA as an effective strategy that targets FOXC2 leading to the resolution of liver fibrosis.

Genome-Wide Characterization of the RNA Exosome Complex in Relation to Growth, Development, and Pathogenicity of Fusarium graminearum.

The RNA exosome complex is a conserved, multisubunit RNase complex that contributes to the processing and degradation of RNAs in mammalian cells. However, the roles of the RNA exosome in phytopathogenic fungi and how it relates to fungal development and pathogenicity remain unclear. Herein, we identified 12 components of the RNA exosome in the wheat fungal pathogen Fusarium graminearum. Live-cell imaging showed that all the components of the RNA exosome complex are localized in the nucleus. FgEXOSC1 and FgEXOSCA were successfully knocked out; they are both involved in the vegetative growth, sexual reproduction, and pathogenicity of F. graminearum. Moreover, deletion of FgEXOSC1 resulted in abnormal toxisomes, decreased deoxynivalenol (DON) production, and downregulation of the expression levels of DON biosynthesis genes. The RNA-binding domain and N-terminal region of FgExosc1 are required for its normal localization and functions. Transcriptome sequencing (RNA-seq) showed that the disruption of FgEXOSC1 resulted in differential expression of 3,439 genes. Genes involved in processing of noncoding RNA (ncRNA), rRNA and ncRNA metabolism, ribosome biogenesis, and ribonucleoprotein complex biogenesis were significantly upregulated. Furthermore, subcellular localization, green fluorescent protein (GFP) pulldown, and coimmunoprecipitation (co-IP) assays demonstrated that FgExosc1 associates with the other components of the RNA exosome to form the RNA exosome complex in F. graminearum. Deletion of FgEXOSC1 and FgEXOSCA reduced the relative expression of some of the other subunits of the RNA exosome. Deletion of FgEXOSC1 affected the localization of FgExosc4, FgExosc6, and FgExosc7. In summary, our study reveals that the RNA exosome is involved in vegetative growth, sexual reproduction, DON production, and pathogenicity of F. graminearum. IMPORTANCE The RNA exosome complex is the most versatile RNA degradation machinery in eukaryotes. However, little is known about how this complex regulates the development and pathogenicity of plant-pathogenic fungi. In this study, we systematically identified 12 components of the RNA exosome complex in Fusarium head blight fungus Fusarium graminearum and first unveiled their subcellular localizations and established their biological functions in relation to the fungal development and pathogenesis. All the RNA exosome components are localized in the nucleus. FgExosc1 and FgExoscA are both required for the vegetative growth, sexual reproduction, DON production and pathogenicity in F. graminearum. FgExosc1 is involved in ncRNA processing, rRNA and ncRNA metabolism process, ribosome biogenesis and ribonucleoprotein complex biogenesis. FgExosc1 associates with the other components of RNA exosome complex and form the exosome complex in F. graminearum. Our study provides new insights into the role of the RNA exosome in regulating RNA metabolism, which is associated with fungal development and pathogenicity.

An integrative approach uncovers transcriptome-wide determinants of mRNA stability regulation in Saccharomyces cerevisiae.

mRNA degradation rate is one of the key stages of gene expression regulation in eukaryotic cells. To date, intertwined processes of post-transcriptional control have been widely investigated, but focused rather on the examination of mechanisms controlling stability of particular protein-coding transcripts. Currently, a wealth of information from structural, biochemical, and high-throughput studies makes it tempting to define general rules governing mRNA stability that could be considered as versatile and valid on a genome-wide scale. Basu et al. analyzed multiple experimental and computational data on Saccharomyces cerevisiae mRNA half-lives as well as on secondary structures and protein-binding sites within transcripts, and collated it with available structures of ribonucleases, that is, enzymes responsible for mRNA degradation. This approach allowed to conclude how particular mRNA features such as lengths of unstructured terminal or internal regions or sequestration into ribonucleoprotein complexes impact half-lives of protein-coding transcripts and to define genome-scale principles of mRNA stability control in yeast.

Post-transcriptional regulation by the exosome complex is required for cell survival and forebrain development via repression of P53 signaling.

Fine-tuned gene expression is crucial for neurodevelopment. The gene expression program is tightly controlled at different levels, including RNA decay. N6-methyladenosine (m6A) methylation-mediated degradation of RNA is essential for brain development. However, m6A methylation impacts not only RNA stability, but also other RNA metabolism processes. How RNA decay contributes to brain development is largely unknown. Here, we show that Exosc10, a RNA exonuclease subunit of the RNA exosome complex, is indispensable for forebrain development. We report that cortical cells undergo overt apoptosis, culminating in cortical agenesis upon conditional deletion of Exosc10 in mouse cortex. Mechanistically, Exosc10 directly binds and degrades transcripts of the P53 signaling-related genes, such as Aen and Bbc3. Overall, our findings suggest a crucial role for Exosc10 in suppressing the P53 pathway, in which the rapid turnover of the apoptosis effectors Aen and Bbc3 mRNAs is essential for cell survival and normal cortical histogenesis.

Nucleolar localization of the yeast RNA exosome subunit Rrp44 hints at early pre-rRNA processing as its main function.

The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast (Saccharomyces cerevisiae) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exosome subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance.

Osteopetrosis-Associated Transmembrane Protein 1 Recruits RNA Exosome To Restrict Hepatitis B Virus Replication.

Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, and 600,000 deaths are caused by HBV-related hepatic failure, liver cirrhosis, and hepatocellular carcinoma annually. It is important to reveal the mechanism underlying the regulation of HBV replication. This study demonstrated that osteopetrosis-associated transmembrane protein 1 (Ostm1) plays an inhibitory role in HBV replication. Ostm1 represses the levels of HBeAg and HBsAg proteins, HBV 3.5-kb and 2.4/2.1-kb RNAs, and core-associated DNA in HepG2, Huh7, and NTCP-HepG2 cells. Notably, Ostm1 has no direct effect on the activity of HBV promoters or the transcription of HBV RNAs; instead, Ostm1 binds to HBV RNA to facilitate RNA decay. Detailed studies further demonstrated that Ostm1 binds to and recruits the RNA exosome complex to promote the degradation of HBV RNAs, and knockdown of the RNA exosome component exonuclease 3 (Exosc3) leads to the elimination of Ostm1-mediated repression of HBV replication. Mutant analyses revealed that the N-terminal domain, the transmembrane domain, and the C-terminal domain are responsible for the repression of HBV replication, and the C-terminal domain is required for interaction with the RNA exosome complex. Moreover, Ostm1 production is not regulated by interferon-α (IFN-α) or IFN-γ, and the expression of IFN signaling components is not affected by Ostm1, suggesting that Ostm1 anti-HBV activity is independent of the IFN signaling pathway. In conclusion, this study revealed a distinct mechanism underlying the repression of HBV replication, in which Ostm1 binds to HBV RNA and recruits RNA exosomes to degrade viral RNA, thereby restricting HBV replication.IMPORTANCE Hepatitis B virus (HBV) is a human pathogen infecting the liver to cause a variety of diseases ranging from acute hepatitis to advanced liver diseases, fulminate hepatitis, liver cirrhosis, and hepatocellular carcinoma, thereby causing a major health problem worldwide. In this study, we demonstrated that Ostm1 plays an inhibitory role in HBV protein production, RNA expression, and DNA replication. However, Ostm1 has no effect on the activities of the four HBV promoters; instead, it binds to HBV RNA and recruits RNA exosomes to promote HBV RNA degradation. We further demonstrated that the anti-HBV activity of Ostm1 is independent of the interferon signaling pathway. In conclusion, this study reveals a distinct mechanism underlying the repression of HBV replication and suggests that Ostm1 is a potential therapeutic agent for HBV infection.

Nuclear sorting of RNA.

The majority of the mammalian genome is transcribed by RNA polymerase II, yielding a vast amount of noncoding RNA (ncRNA) in addition to the standard production of mRNA. The typical nuclear biogenesis of mRNA relies on the tightly controlled coupling of co- and post-transcriptional processing events, which ultimately results in the export of transcripts into the cytoplasm. These processes are subject to surveillance by nuclear RNA decay pathways to prevent the export of aberrant, or otherwise "non-optimal," transcripts. However, unlike mRNA, many long ncRNAs are nuclear retained and those that maintain enduring functions must employ precautions to evade decay. Proper sorting and localization of RNA is therefore an essential activity in eukaryotic cells and the formation of ribonucleoprotein complexes during early stages of RNA synthesis is central to deciding such transcript fate. This review details our current understanding of the pathways and factors that direct RNAs towards a particular destiny and how transcripts combat the adverse conditions of the nucleus. This article is categorized under: RNA Export and Localization > Nuclear Export/Import RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Comparative Poly(A)+ RNA Interactome Capture of RNA Surveillance Mutants.

RNA exosome complexes degrade many different RNA substrates. Substrate selection and targeting to the exosome complex rely on cofactors, which bind to the substrate RNA, recruit the exosome complex, and help to remodel the associated ribonucleoprotein particle to facilitate RNA degradation. These cofactors are RNA-binding proteins, but their interaction with RNA may be very transient because the RNAs they are bound to are rapidly turned over by the exosome complex. Hence, the cofactors involved in the degradation of many exosome substrates are unknown. Here, we describe comparative poly(A)+ RNA interactome capture as a method to screen for novel RNA-binding proteins involved in exosome-dependent RNA decay.For this, we compare the poly(A)+ RNA interactome of wild-type cells to that of RNA surveillance mutants, where the decay of exosome substrates is compromised and occupancy of exosome cofactors on RNA is strongly increased. More specifically, protein-RNA complexes in wild-type and mutant cells are UV-cross-linked in vivo after labeling with the photoactivatable nucleoside analogue 4-thiouracil. Following cell lysis, protein-RNA complexes are selected on oligo d(T) beads, subjected to stringent washes, and eluted in a low salt buffer. After RNase digestion of cross-linked RNA, RNA-binding proteins that are enriched in the mutant samples are identified by quantitative mass spectrometry. Here, we quantitatively compare the RNA-protein interactomes of wild-type and rrp6Δ cells to selectively determine cofactors of the nuclear RNA exosome complex in fission yeast. With minor modifications, the comparative interactome approach can easily be adapted to study a range of different RNA-dependent processes in various cellular systems.

Native Mass Spectrometry Analysis of Affinity-Captured Endogenous Yeast RNA Exosome Complexes.

Native mass spectrometry (MS) enables direct mass measurement of intact protein assemblies generating relevant subunit composition and stoichiometry information. Combined with cross-linking and structural data, native MS-derived information is crucial for elucidating the architecture of macromolecular assemblies by integrative structural methods. The exosome complex from budding yeast was among the first endogenous protein complexes to be affinity isolated and subsequently characterized by this technique, providing improved understanding of its composition and structure. We present a protocol that couples efficient affinity capture of yeast exosome complexes and sensitive native MS analysis, including rapid affinity isolation of the endogenous exosome complex from cryolysed yeast cells, elution in nondenaturing conditions by protease cleavage, depletion of the protease, buffer exchange, and native MS measurements using an Orbitrap-based instrument (Exactive Plus EMR).

Chemical Cross-Linking and Mass Spectrometric Analysis of the Endogenous Yeast Exosome Complexes.

Chemical cross-linking and mass spectrometric readout (CX-MS) has become a useful toolkit for structural analysis of protein complexes. CX-MS enables rapid detection of a larger number of cross-link peptides from the chemically cross-linked protein assembly, providing invaluable cross-link spatial restraints to understand the architecture of the complex. Since CX-MS is complementary with other structural and computational modeling tools, it can be used for integrative structural determination of large native protein assemblies. However, due to technical limitations, current CX-MS applications have still been predominantly confined to complexes reconstituted from recombinant proteins where large amount of purified materials are available. Cross-linking and hybrid structural proteomic analysis of endogenous protein complexes remains a challenge. In this chapter, we present a protocol that efficiently couples affinity capture of endogenous complexes with sensitive CX-MS analysis, with particular application to the yeast RNA processing exosome complexes.

Exosome complex genes mediate RNA degradation and predict survival in mantle cell lymphoma.

Exosome complex (EXOSC) genes, which encode a multi-protein intracellular complex, mediate the degradation of various types of RNA molecules. EXOSCs, also known as polymyositis/scleroderma complexes, exist in eukaryotic cells and archaea, and primarily mediate 3' to 5'mRNA degradation. However, how EXOSC genes are implicated in processes of B-cell immune-associated pathways and B-cell tumorigenesis remains unclear. The present bioinformatics study indicated that 6 of 10 EXOSC genes, particularly the EXO.index, were able to predict the survival of patients with mantle cell lymphoma (MCL), by analyzing gene expression profiles of 123 patients with MCL from the Gene Expression Omnibus database. The results suggested that EXOSC gene expression may be a molecular marker for MCL. Compared with the whole transcript profile, patients with MCL with a high EXO.index exhibited poorer survival and decreased RNA levels, which was also verified in a second dataset. The EXOSC genes may be associated with DNA repair and B-cell activation pathways, which may be the cause of poorer survival of patients with MCL.

The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.

Eukaryotic ribosomal biogenesis is a high-energy-demanding and complex process that requires hundreds of trans-acting factors to dynamically build the highly-organized 40S and 60S subunits. Each ribonucleoprotein complex comprises specific rRNAs and ribosomal proteins that are organized into functional domains. The RNA exosome complex plays a crucial role as one of the pre-60S-processing factors, because it is the RNase responsible for processing the 7S pre-rRNA to the mature 5.8S rRNA. The yeast pre-60S assembly factor Nop53 has previously been shown to associate with the nucleoplasmic pre-60S in a region containing the "foot" structure assembled around the 3' end of the 7S pre-rRNA. Nop53 interacts with 25S rRNA and with several 60S assembly factors, including the RNA exosome, specifically, with its catalytic subunit Rrp6 and with the exosome-associated RNA helicase Mtr4. Nop53 is therefore considered the adaptor responsible for recruiting the exosome complex for 7S processing. Here, using proteomics-based approaches in budding yeast to analyze the effects of Nop53 on the exosome interactome, we found that the exosome binds pre-ribosomal complexes early during the ribosome maturation pathway. We also identified interactions through which Nop53 modulates exosome activity in the context of 60S maturation and provide evidence that in addition to recruiting the exosome, Nop53 may also be important for positioning the exosome during 7S processing. On the basis of these findings, we propose that the exosome is recruited much earlier during ribosome assembly than previously thought, suggesting the existence of additional interactions that remain to be described.

Stress-induced nuclear depletion of Entamoeba histolytica 3'-5' exoribonuclease EhRrp6 and its role in growth and erythrophagocytosis.

The 3'-5' exoribonuclease Rrp6 is a key enzyme in RNA homeostasis involved in processing and degradation of many stable RNA precursors, aberrant transcripts, and noncoding RNAs. We previously have shown that in the protozoan parasite Entamoeba histolytica, the 5'-external transcribed spacer fragment of pre-rRNA accumulates under serum starvation-induced growth stress. This fragment is a known target of degradation by Rrp6. Here, we computationally and biochemically characterized EhRrp6 and found that it contains the catalytically important EXO and HRDC domains and exhibits exoribonuclease activity with both unstructured and structured RNA substrates, which required the conserved DEDD-Y catalytic-site residues. It lacked the N-terminal PMC2NT domain for binding of the cofactor Rrp47, but could functionally complement the growth defect of a yeast rrp6 mutant. Of note, no Rrp47 homologue was detected in E. histolytica Immunolocalization studies revealed that EhRrp6 is present both in the nucleus and cytosol of normal E. histolytica cells. However, growth stress induced its complete loss from the nuclei, reversed by proteasome inhibitors. EhRrp6-depleted E. histolytica cells were severely growth restricted, and EhRrp6 overexpression protected the cells against stress, suggesting that EhRrp6 functions as a stress sensor. Importantly EhRrp6 depletion reduced erythrophagocytosis, an important virulence determinant of E. histolytica This reduction was due to a specific decrease in transcript levels of some phagocytosis-related genes (Ehcabp3 and Ehrho1), whereas expression of other genes (Ehcabp1, Ehcabp6, Ehc2pk, and Eharp2/3) was unaffected. This is the first report of the role of Rrp6 in cell growth and stress responses in a protozoan parasite.

RNA Exosome Complex-Mediated Control of Redox Status in Pluripotent Stem Cells.

The RNA exosome complex targets AU-rich element (ARE)-containing mRNAs in eukaryotic cells. We identified a transcription factor, ZSCAN10, which binds to the promoters of multiple RNA exosome complex subunits in pluripotent stem cells to maintain subunit gene expression. We discovered that induced pluripotent stem cell clones generated from aged tissue donors (A-iPSC) show poor expression of ZSCAN10, leading to poor RNA exosome complex expression, and a subsequent elevation in ARE-containing RNAs, including glutathione peroxidase 2 (Gpx2). Excess GPX2 leads to excess glutathione-mediated reactive oxygen species scavenging activity that blunts the DNA damage response and apoptosis. Expression of ZSCAN10 in A-iPSC recovers RNA exosome gene expression, the DNA damage response, and apoptosis. These findings reveal the central role of ZSCAN10 and the RNA exosome complex in maintaining pluripotent stem cell redox status to support a normal DNA damage response.

Recessive mutation in EXOSC3 associates with mitochondrial dysfunction and pontocerebellar hypoplasia.

Recessive mutations in EXOSC3, encoding a subunit of the human RNA exosome complex, cause pontocerebellar hypoplasia type 1b (PCH1B). We report a boy with severe muscular hypotonia, psychomotor retardation, progressive microcephaly, and cerebellar atrophy. Biochemical abnormalities comprised mitochondrial complex I and pyruvate dehydrogenase complex (PDHc) deficiency. Whole exome sequencing uncovered a known EXOSC3 mutation p.(D132A) as the underlying cause. In patient fibroblasts, a large portion of the EXOSC3 protein was trapped in the cytosol. MtDNA copy numbers in muscle were reduced to 35%, but mutations in the mtDNA and in nuclear mitochondrial genes were ruled out. RNA-Seq of patient muscle showed highly increased mRNA copy numbers, especially for genes encoding structural subunits of OXPHOS complexes I, III, and IV, possibly due to reduced degradation by a dysfunctional exosome complex. This is the first case of mitochondrial dysfunction associated with an EXOSC3 mutation, which expands the phenotypic spectrum of PCH1B. We discuss the links between exosome and mitochondrial dysfunction.

Identification of karyopherins involved in the nuclear import of RNA exosome subunit Rrp6 in Saccharomyces cerevisiae.

The exosome is a conserved multiprotein complex essential for RNA processing and degradation. The nuclear exosome is a key factor for pre-rRNA processing through the activity of its catalytic subunits, Rrp6 and Rrp44. In Saccharomyces cerevisiae, Rrp6 is exclusively nuclear and has been shown to interact with exosome cofactors. With the aim of analyzing proteins associated with the nuclear exosome, in this work, we purified the complex with Rrp6-TAP, identified the co-purified proteins by mass spectrometry, and found karyopherins to be one of the major groups of proteins enriched in the samples. By investigating the biological importance of these protein interactions, we identified Srp1, Kap95, and Sxm1 as the most important karyopherins for Rrp6 nuclear import and the nuclear localization signals recognized by them. Based on the results shown here, we propose a model of multiple pathways for the transport of Rrp6 to the nucleus.

RNA Exosome Complex Regulates Stability of the Hepatitis B Virus X-mRNA Transcript in a Non-stop-mediated (NSD) RNA Quality Control Mechanism.

Hepatitis B virus (HBV) is a stealth virus, minimally inducing the interferon system required for efficient induction of both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of other, interferon-independent pathways leading to viral clearance. Given the known ability of helicases to bind viral nucleic acids, we performed a functional screening assay to identify helicases that regulate HBV replication. We identified the superkiller viralicidic activity 2-like (SKIV2L) RNA helicase (a homolog of the Saccharomyces cerevisiae Ski2 protein) on the basis of its direct and preferential interaction with HBV X-mRNA. This interaction was essential for HBV X-mRNA degradation at the RNA exosome. The degradation of HBV X-mRNA at the RNA exosome was also mediated by HBS1L (HBS1-like translational GTPase) protein, a known component of the host RNA quality control system. We found that the redundant HBV-precore translation initiation site present at the 3'-end of HBV X-mRNA (3' precore) is translationally active. The initiation of translation from this site without a proper stop codon was identified by the non-stop-mediated RNA decay mechanism leading to its degradation. Although 3' precore is present in the five main HBV-RNA transcripts, only X-mRNA lacks the presence of an upstream start codons for large, middle, and small (L, M, and S) HBV surface proteins. These upstream codons are in-frame with 3' precore translation initiation site, blocking its translation from the other HBV-mRNA transcripts. To our knowledge, this is the first demonstration of the anti-viral function of the non-stop-mediated RNA decay mechanism.

Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.

Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.

Exosome Cofactors Connect Transcription Termination to RNA Processing by Guiding Terminated Transcripts to the Appropriate Exonuclease within the Nuclear Exosome.

The yeast Nrd1 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNApII) through its CTD-interacting domain (CID) and also associates with the nuclear exosome, thereby acting as both a transcription termination and RNA processing factor. Previously, we found that the Nrd1 CID is required to recruit the nuclear exosome to the Nrd1 complex, but it was not clear which exosome subunits were contacted. Here, we show that two nuclear exosome cofactors, Mpp6 and Trf4, directly and competitively interact with the Nrd1 CID and differentially regulate the association of Nrd1 with two catalytic subunits of the exosome. Importantly, Mpp6 promotes the processing of Nrd1-terminated transcripts preferentially by Dis3, whereas Trf4 leads to Rrp6-dependent processing. This suggests that Mpp6 and Trf4 may play a role in choosing a particular RNA processing route for Nrd1-terminated transcripts within the exosome by guiding the transcripts to the appropriate exonuclease.