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Elena Conti - One of the best experts on this subject based on the ideXlab platform.
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distinct and evolutionary conserved structural features of the human nuclear Exosome Complex
eLife, 2018Co-Authors: Piotr Gerlach, Jan M Schuller, Fabien Bonneau, Jerome Basquin, Peter Reichelt, Sebastian Falk, Elena ContiAbstract:The nuclear RNA Exosome Complex mediates the processing of structured RNAs and the decay of aberrant non-coding RNAs, an important function particularly in human cells. Most mechanistic studies to date have focused on the yeast system. Here, we reconstituted and studied the properties of a recombinant 14-subunit human nuclear Exosome Complex. In biochemical assays, the human Exosome embeds a longer RNA channel than its yeast counterpart. The 3.8 A resolution cryo-EM structure of the core Complex bound to a single-stranded RNA reveals that the RNA channel path is formed by two distinct features of the hDIS3 exoribonuclease: an open conformation and a domain organization more similar to bacterial RNase II than to yeast Rrp44. The cryo-EM structure of the holo-Complex shows how obligate nuclear cofactors position the hMTR4 helicase at the entrance of the core Complex, suggesting a striking structural conservation from lower to higher eukaryotes.
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structure of the nuclear Exosome captured on a maturing preribosome
Science, 2018Co-Authors: Jan M Schuller, Sebastian Falk, Lisa Fromm, Ed Hurt, Elena ContiAbstract:The RNA Exosome Complex processes and degrades a wide range of transcripts, including ribosomal RNAs (rRNAs). We used cryo–electron microscopy to visualize the yeast nuclear Exosome holoComplex captured on a precursor large ribosomal subunit (pre-60 S ) during 7 S -to-5.8 S rRNA processing. The cofactors of the nuclear Exosome are sandwiched between the ribonuclease core Complex (Exo-10) and the remodeled “foot” structure of the pre-60 S particle, which harbors the 5.8 S rRNA precursor. The Exosome-associated helicase Mtr4 recognizes the preribosomal substrate by docking to specific sites on the 25 S rRNA, captures the 3′ extension of the 5.8 S rRNA, and channels it toward Exo-10. The structure elucidates how the Exosome forms a structural and functional unit together with its massive pre-60 S substrate to process rRNA during ribosome maturation.
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structure of a cytoplasmic 11 subunit rna Exosome Complex
Molecular Cell, 2016Co-Authors: Eva Kowalinski, Peter Reichelt, Alexander Kogel, Judith Ebert, Elisabeth Stegmann, Bianca Habermann, Elena ContiAbstract:The RNA Exosome Complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the Exosome (Exo10) functions together with the conserved Ski Complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 A resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this Complex, pointing to Ski7 as a constitutive component of the cytosolic Exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast Complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like Exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor.
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molecular mechanism of processive 3 to 5 rna translocation in the active subunit of the rna Exosome Complex
Journal of the American Chemical Society, 2016Co-Authors: Lela Vukovic, Debora L Makino, Elena Conti, Christophe Chipot, Klaus SchultenAbstract:Recent experimental studies revealed structural details of 3' to 5' degradation of RNA molecules, performed by the Exosome Complex. ssRNA is channeled through its multisubunit ring-like core into the active site tunnel of its key exonuclease subunit Rrp44, which acts both as an enzyme and a motor. Even in isolation, Rrp44 can pull and sequentially cleave RNA nucleotides, one at a time, without any external energy input and release a final 3-5 nucleotide long product. Using molecular dynamics simulations, we identify the main factors that control these processes. Our free energy calculations reveal that RNA transfer from solution into the active site of Rrp44 is highly favorable, but dependent on the length of the RNA strand. While RNA strands formed by 5 nucleotides or more correspond to a decreasing free energy along the translocation coordinate toward the cleavage site, a 4-nucleotide RNA experiences a free energy barrier along the same direction, potentially leading to incomplete cleavage of ssRNA and the release of short (3-5) nucleotide products. We provide new insight into how Rrp44 catalyzes a localized enzymatic reaction and performs an action distributed over several RNA nucleotides, leading eventually to the translocation of whole RNA segments into the position suitable for cleavage.
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rna degradation paths in a 12 subunit nuclear Exosome Complex
Nature, 2015Co-Authors: Debora L Makino, Elisabeth Stegmann, Benjamin Schuch, Marc Baumgartner, Claire Basquin, Elena ContiAbstract:The eukaryotic Exosome is a conserved RNA-degrading Complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear Exosome containing the 9-subunit core, the 3'-5' RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in Complex with different RNAs. The combined structural and biochemical data of this 12-subunit Complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6-Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits.
David Tollervey - One of the best experts on this subject based on the ideXlab platform.
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transcriptome wide analysis of alternative routes for rna substrates into the Exosome Complex
PLOS Genetics, 2017Co-Authors: Clementine Delanforino, Claudia Schneider, David TollerveyAbstract:The RNA Exosome Complex functions in both the accurate processing and rapid degradation of many classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the Exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway in vivo. We used CRAC (UV crosslinking and analysis of cDNA) in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear Exosome-cofactor Mtr4 and with individual Exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We compared Exosome Complexes lacking Rrp44 exonuclease activity, carrying a mutation in the Rrp44 S1 RNA-binding domain predicted to disfavor direct access, or with multiple mutations in Rrp41 reported to impede RNA access to the central channel in vitro. Preferential use of channel-threading was seen for mRNAs, 5S rRNA, scR1 (SRP) and aborted tRNAs transcripts. Conversely, pre-tRNAs preferentially accessed Rrp44 directly. Both routes participated in degradation and maturation of RNAPI transcripts, with hand-over during processing. Rrp41 mutations blocked substrate passage through the channel to Rrp44 only for cytoplasmic mRNAs, supporting the predicted widening of the lumen in the Rrp6-associated, nuclear Complex. Many Exosome substrates exhibited clear preferences for a specific path to Rrp44. Other targets showed redundancy, possibly allowing the efficient handling of highly diverse RNA-protein Complexes and RNA structures. Both threading and direct access routes involve the RNA helicase Mtr4. mRNAs that are predominately nuclear or cytoplasmic Exosome substrates can be distinguished in vivo.
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rna quality control by the Exosome
Nature Reviews Molecular Cell Biology, 2006Co-Authors: Jonathan Houseley, John Lacava, David TollerveyAbstract:The Exosome Complex of 3'-->5' exonucleases is an important component of the RNA-processing machinery in eukaryotes. This Complex functions in the accurate processing of nuclear RNA precursors and in the degradation of RNAs in both the nucleus and the cytoplasm. However, it has been unclear how different classes of substrate are distinguished from one another. Recent studies now provide insights into the regulation and structure of the Exosome, and they reveal striking similarities between the process of RNA degradation in bacteria and eukaryotes.
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RNA degradation by the Exosome is promoted by a nuclear polyadenylation Complex
Cell, 2005Co-Authors: John Lacava, Jonathan Houseley, Cosmin Saveanu, Elisabeth Petfalski, Elizabeth Thompson, Alain Jacquier, David TollerveyAbstract:The Exosome Complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein Complexes in vivo. However, the purified Exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation Complex containing a known Exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation Complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the Exosome suppressed poly(A) tail addition, while TRAMP stimulated Exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized Exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.
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musing on the structural organization of the Exosome Complex
Nature Structural & Molecular Biology, 2000Co-Authors: Philip Mitchell, David TollerveyAbstract:The Exosome Complex of 3′→5′ exoribonucleases functions in both the precise processing of 3′ extended precursor molecules to mature stable RNAs and the complete degradation of other RNAs. Both processing and degradative activities of the Exosome depend on additional cofactors, notably the putative RNA helicases Mtr4p and Ski2p. It is not known how these factors regulate Exosome function or how the Exosome distinguishes RNAs destined for processing events from substrates that are to be completely degraded. Here we review the available data concerning the modes of action of the Exosome and relate these to possible structural arrangements for the Complex. As no detailed structural data are yet available for the Exosome Complex, or any of its constituent enzymes, this discussion will rely heavily on rather speculative models.
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precursors to the u3 small nucleolar rna lack small nucleolar rnp proteins but are stabilized by la binding
Molecular and Cellular Biology, 2000Co-Authors: Joanna Kufel, Elisabeth Petfalski, Christine Allmang, Guillaume Chanfreau, Denis L J Lafontaine, David TollerveyAbstract:Almost all small eukaryotic RNAs are processed from transiently stabilized 3'-extended forms. A key question is how and why such intermediates are stabilized and how they can then be processed to the mature RNA. Here we report that yeast U3 is also processed from a 3'-extended precursor. The major 3'-extended forms of U3 (U3-3'I and -II) lack the cap trimethylation present in mature U3 and are not associated with small nucleolar RNP (snoRNP) proteins that bind mature U3, i.e., Nop1p, Nop56p, and Nop58p. Depletion of Nop58p leads to the loss of mature U3 but increases the level of U3-3'I and -II, indicating a requirement for the snoRNP proteins for final maturation. Pre-U3 is cleaved by the endonuclease Rnt1p, but U3-3'I and -II do not extend to the Rnt1p cleavage sites. Rather, they terminate at poly(U) tracts, suggesting that they might be bound by Lhp1p (the yeast homologue of La). Immunoprecipitation of Lhp1p fused to Staphylococcus aureus protein A resulted in coprecipitation of both U3-3'I and -II. Deletion of LHP1, which is nonessential, led to the loss of U3-3'I and -II. We conclude that pre-U3 is cleaved by Rnt1p, followed by exonuclease digestion to U3-3'I and -II. These species are stabilized against continued degradation by binding of Lhp1p. Displacement of Lhp1p by binding of the snoRNP proteins allows final maturation, which involves the Exosome Complex of 3'-->5' exonucleases.
Carla C Oliveira - One of the best experts on this subject based on the ideXlab platform.
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the ribosome assembly factor nop53 controls association of the rna Exosome with pre 60s particles in yeast
Journal of Biological Chemistry, 2019Co-Authors: Leidy Paola Paez Cepeda, Felipe F M Bagatelli, Renata M Santos, Marlon D M Santos, Fabio C S Nogueira, Carla C OliveiraAbstract: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.
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differential expression of rna Exosome subunits in the amphibian lithobates catesbeianus during reproductive and non reproductive periods
BMC Research Notes, 2019Co-Authors: Breno Henrique Caneguim, M M Santoni, Estela Sassocerri, André Baggio, Sandro Roberto Valentini, Caren C Helbing, Carla C OliveiraAbstract:The RNA Exosome is an evolutionarily conserved 3′–5′ exoribonucleolytic protein Complex involved in processing and degradation of different classes of nuclear and cytoplasmic RNAs, and, therefore, important for the posttranscriptional control of gene expression. Despite the extensive in vivo functional studies and the structural data on the RNA Exosome, few studies have been performed on the localization and expression of Exosome subunits during gametogenesis, process during which gene expression is largely controlled at the posttranscriptional level. We report the identification of Exosome subunits in Lithobates catesbeianus and analysis of the differential subcellular localization of RNA Exosome core and catalytic subunits in testis cells. In addition, we show seasonal differences in the expression levels of four Exosome subunits in different organs. In addition to being part of the RNA Exosome Complex, its subunits might participate independently of the Complex in the control of gene expression during seasonal variation in bullfrog tissues. These results may be relevant for other eukaryotic species.
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Differential expression of RNA Exosome subunits in the amphibian Lithobates catesbeianus during reproductive and non-reproductive periods
'Springer Science and Business Media LLC', 2019Co-Authors: J. S. Luz, Breno Henrique Caneguim, M M Santoni, André Baggio, Sandro Roberto Valentini, Caren C Helbing, E. Sasso-cerri, Carla C OliveiraAbstract:Abstract Objective The RNA Exosome is an evolutionarily conserved 3′–5′ exoribonucleolytic protein Complex involved in processing and degradation of different classes of nuclear and cytoplasmic RNAs, and, therefore, important for the posttranscriptional control of gene expression. Despite the extensive in vivo functional studies and the structural data on the RNA Exosome, few studies have been performed on the localization and expression of Exosome subunits during gametogenesis, process during which gene expression is largely controlled at the posttranscriptional level. Results We report the identification of Exosome subunits in Lithobates catesbeianus and analysis of the differential subcellular localization of RNA Exosome core and catalytic subunits in testis cells. In addition, we show seasonal differences in the expression levels of four Exosome subunits in different organs. In addition to being part of the RNA Exosome Complex, its subunits might participate independently of the Complex in the control of gene expression during seasonal variation in bullfrog tissues. These results may be relevant for other eukaryotic species
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the pyrococcus Exosome Complex structural and functional characterization
Journal of Biological Chemistry, 2006Co-Authors: Celso Raul Romero Ramos, Cristiano L P Oliveira, I Torriani, Carla C OliveiraAbstract:The Exosome is a conserved eukaryotic enzymatic Complex that plays an essential role in many pathways of RNA processing and degradation. Here, we describe the structural characterization of the predicted archaeal Exosome in solution using small angle x-ray scattering. The structure model calculated from the small angle x-ray scattering pattern provides an indication of the existence of a disk-shaped structure, corresponding to the "RNases PH ring" Complex formed by the proteins aRrp41 and aRrp42. The RNases PH ring Complex corresponds to the core of the Exosome, binds RNA, and has phosphorolytic and polymerization activities. Three additional molecules of the RNA-binding protein aRrp4 are attached to the core as extended and flexible arms that may direct the substrates to the active sites of the Exosome. In the presence of aRrp4, the activity of the core Complex is enhanced, suggesting a regulatory role for this protein. The results shown here also indicate the participation of the Exosome in RNA metabolism in Archaea, as was established in Eukarya.
Debora L Makino - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of processive 3 to 5 rna translocation in the active subunit of the rna Exosome Complex
Journal of the American Chemical Society, 2016Co-Authors: Lela Vukovic, Debora L Makino, Elena Conti, Christophe Chipot, Klaus SchultenAbstract:Recent experimental studies revealed structural details of 3' to 5' degradation of RNA molecules, performed by the Exosome Complex. ssRNA is channeled through its multisubunit ring-like core into the active site tunnel of its key exonuclease subunit Rrp44, which acts both as an enzyme and a motor. Even in isolation, Rrp44 can pull and sequentially cleave RNA nucleotides, one at a time, without any external energy input and release a final 3-5 nucleotide long product. Using molecular dynamics simulations, we identify the main factors that control these processes. Our free energy calculations reveal that RNA transfer from solution into the active site of Rrp44 is highly favorable, but dependent on the length of the RNA strand. While RNA strands formed by 5 nucleotides or more correspond to a decreasing free energy along the translocation coordinate toward the cleavage site, a 4-nucleotide RNA experiences a free energy barrier along the same direction, potentially leading to incomplete cleavage of ssRNA and the release of short (3-5) nucleotide products. We provide new insight into how Rrp44 catalyzes a localized enzymatic reaction and performs an action distributed over several RNA nucleotides, leading eventually to the translocation of whole RNA segments into the position suitable for cleavage.
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rna degradation paths in a 12 subunit nuclear Exosome Complex
Nature, 2015Co-Authors: Debora L Makino, Elisabeth Stegmann, Benjamin Schuch, Marc Baumgartner, Claire Basquin, Elena ContiAbstract:The eukaryotic Exosome is a conserved RNA-degrading Complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear Exosome containing the 9-subunit core, the 3'-5' RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in Complex with different RNAs. The combined structural and biochemical data of this 12-subunit Complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6-Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits.
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molecular mechanism of processive 3 to 5 rna translocation in the rna Exosome Complex
Biophysical Journal, 2015Co-Authors: Lela Vukovic, Debora L Makino, Elena Conti, Christophe Chipot, Klaus SchultenAbstract:3’ to 5’ degradation of a wide range of RNA molecules is performed by the Exosome Complex, as a key part of cellular quality control. Recent structural studies of this Complex revealed that ssRNA is channeled through its multisubunit ring-like core into the active site tunnel of its exonuclease subunit Rrp44. Rrp44, both alone and in the Exosome, processively cleaves RNA nucleotides one at a time, without consuming the energy of ATP, and releases a final 3-5 nucleotide product. We use molecular dynamics simulations and free energy calculations to identify the factors that enable processivity of RNA translocation and cleavage in the Exosome Complex. Our simulations reveal large and favorable free energies of RNA transfer from solution into the active site of Rrp44. The free energy profiles that characterize RNA translocation within the active site of Rrp44 are found to be dependent on the length of the RNA strand. While RNA strands formed by 5 nucleotides or more have downhill free energy profiles along the translocation coordinate towards the cleavage site, a 4-nucleotide RNA has a free energy barrier along the same coordinate, potentially leading to incomplete cleavage of ssRNA and the release of short (3-5) nucleotide products. Furthermore, dynamic insights gained from the performed simulations help elucidate the concerted nature of RNA translocation through the Exosome Complex.
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structure determination of an 11 subunit Exosome in Complex with rna by molecular replacement
Acta Crystallographica Section D-biological Crystallography, 2013Co-Authors: Debora L Makino, Elena ContiAbstract:The RNA Exosome is an evolutionarily conserved multi-protein Complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the Exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the Exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subComplexes of eukaryotic Exosomes or related prokaryotic Exosome-like Complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa Exosome Complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.
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crystal structure of an rna bound 11 subunit eukaryotic Exosome Complex
Nature, 2013Co-Authors: Debora L Makino, Marc Baumgartner, Elena ContiAbstract:The Exosome is the major 3'-5' RNA-degradation Complex in eukaryotes. The ubiquitous core of the yeast Exosome (Exo-10) is formed by nine catalytically inert subunits (Exo-9) and a single active RNase, Rrp44. In the nucleus, the Exo-10 core recruits another nuclease, Rrp6. Here we crystallized an approximately 440-kilodalton Complex of Saccharomyces cerevisiae Exo-10 bound to a carboxy-terminal region of Rrp6 and to an RNA duplex with a 3'-overhang of 31 ribonucleotides. The 2.8 A resolution structure shows how RNA is funnelled into the Exo-9 channel in a single-stranded conformation by an unwinding pore. Rrp44 adopts a closed conformation and captures the RNA 3'-end that exits from the side of Exo-9. Exo-9 subunits bind RNA with sequence-unspecific interactions reminiscent of archaeal Exosomes. The substrate binding and channelling mechanisms of 3'-5' RNA degradation Complexes are conserved in all kingdoms of life.
Emery H Bresnick - One of the best experts on this subject based on the ideXlab platform.
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rna regulatory Exosome Complex confers cellular survival to promote erythropoiesis
Nucleic Acids Research, 2021Co-Authors: Charu Mehta, Isabela Fraga De Andrade, Daniel R Matson, Colin N Dewey, Emery H BresnickAbstract:Cellular differentiation requires vast remodeling of transcriptomes, and therefore machinery mediating remodeling controls differentiation. Relative to transcriptional mechanisms governing differentiation, post-transcriptional processes are less well understood. As an important post-transcriptional determinant of transcriptomes, the RNA Exosome Complex (EC) mediates processing and/or degradation of select RNAs. During erythropoiesis, the erythroid transcription factor GATA1 represses EC subunit genes. Depleting EC structural subunits prior to GATA1-mediated repression is deleterious to erythroid progenitor cells. To assess the importance of the EC catalytic subunits Dis3 and Exosc10 in this dynamic process, we asked if these subunits function non-redundantly to control erythropoiesis. Dis3 or Exosc10 depletion in primary murine hematopoietic progenitor cells reduced erythroid progenitors and their progeny, while sparing myeloid cells. Dis3 loss severely compromised erythroid progenitor and erythroblast survival, rendered erythroblasts hypersensitive to apoptosis-inducing stimuli and induced γ-H2AX, indicative of DNA double-stranded breaks. Dis3 loss-of-function phenotypes were more severe than those caused by Exosc10 depletion. We innovated a genetic rescue system to compare human Dis3 with multiple myeloma-associated Dis3 mutants S447R and R750K, and only wild type Dis3 was competent to rescue progenitors. Thus, Dis3 establishes a disease mutation-sensitive, cell type-specific survival mechanism to enable a differentiation program.
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post transcriptional control of cellular differentiation by the rna Exosome Complex
Nucleic Acids Research, 2020Co-Authors: Isabela Fraga De Andrade, Charu Mehta, Emery H BresnickAbstract:Given the Complexity of intracellular RNA ensembles and vast phenotypic remodeling intrinsic to cellular differentiation, it is instructive to consider the role of RNA regulatory machinery in controlling differentiation. Dynamic post-transcriptional regulation of protein-coding and non-coding transcripts is vital for establishing and maintaining proteomes that enable or oppose differentiation. By contrast to extensively studied transcriptional mechanisms governing differentiation, many questions remain unanswered regarding the involvement of post-transcriptional mechanisms. Through its catalytic activity to selectively process or degrade RNAs, the RNA Exosome Complex dictates the levels of RNAs comprising multiple RNA classes, thereby regulating chromatin structure, gene expression and differentiation. Although the RNA Exosome would be expected to control diverse biological processes, studies to elucidate its biological functions and how it integrates into, or functions in parallel with, cell type-specific transcriptional mechanisms are in their infancy. Mechanistic analyses have demonstrated that the RNA Exosome confers expression of a differentiation regulatory receptor tyrosine kinase, downregulates the telomerase RNA component TERC, confers genomic stability and promotes DNA repair, which have considerable physiological and pathological implications. In this review, we address how a broadly operational RNA regulatory Complex interfaces with cell type-specific machinery to control cellular differentiation.
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Exosome Complex orchestrates developmental signaling to balance proliferation and differentiation during erythropoiesis
eLife, 2016Co-Authors: Skye C Mciver, Yoona Kang, David T Yang, Koichi R Katsumura, Elsa Davids, Peng Liu, Emery H BresnickAbstract:Since the highly conserved Exosome Complex mediates the degradation and processing of multiple classes of RNAs, it almost certainly controls diverse biological processes. How this post-transcriptional RNA-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that Exosome Complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in Mus musculus, we discovered that the Exosome Complex is a vital determinant of a developmental signaling transition that dictates proliferation/amplification versus differentiation. Exosome Complex integrity in erythroid precursor cells ensures Kit receptor tyrosine kinase expression and stem cell factor/Kit signaling, while preventing responsiveness to erythropoietin-instigated signals that promote differentiation. Functioning as a gatekeeper of this developmental signaling transition, the Exosome Complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts.
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the rna degrading Exosome Complex is an endogenous suppressor of erythroid maturation
Blood, 2014Co-Authors: Skye C Mciver, Yoona Kang, Andrew W Devilbiss, Chelsea A Odriscoll, David T Yang, Saghi Ghaffari, Emery H BresnickAbstract:Abstract Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. We demonstrated that the master regulator of erythropoiesis, GATA-1, induces expression of genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. We conducted studies to assess whether the GATA-1/Foxo3 cooperativity is restricted to the control of autophagy, or if it more broadly impacts the erythroid cell transcriptome. Analysis of the GATA-1/Foxo3-dependent transcriptome in erythroid cells revealed a target gene ensemble extending beyond autophagy, but representing only a small fraction of the Complex GATA-1-dependent target gene ensemble. GATA-1/Foxo3 repressed expression of genes encoding two Exosome Complex components, Exosc5 and Exosc8. The Exosome Complex functions in one of the major RNA degradation pathways in diverse cell types, mediates splicing and degradation of mRNAs and non-coding RNAs, and functions in epigenetic gene regulation. As the role of the Exosome Complex in erythropoiesis, and more broadly in hematopoiesis, had not been described previously, we conducted biological and mechanistic studies to determine whether the endogenous Exosome Complex has important roles in the development and/or function of erythroid cells. Strikingly, downregulating expression of endogenous Exosome components, Exosc8, Exosc9 and the catalytic component Dis3 dramatically increased the percentage of primary erythroid precursor cells in the R4 (polychromatic and orthrochromatic orthrochromatic) population from 1% in control cells to 30%, 28% and 16% respectively. We have extended these initial findings to explore key mechanistic and biological questions. Using the Exosome Complex high-resolution crystal structure as a guide, we are conducting loss-of-function studies to establish whether additional Exosome Complex components that serve structural roles in the Complex (Exosc4 and Exosc7), bind RNA substrates (Exosc1), and degrade RNAs (Dis3L and Exosc10) are also important determinants of erythroid maturation. Initial studies indicate that multiple components suppress maturation, but differ quantitatively in their importance. Studies are underway to test the hypothesis that downregulating Exosc8 or Exosc9 severely disrupts the integrity of the Exosome Complex, whereas certain other components are less critical for Complex integrity and function. We have developed a co-immunoprecipitation assay to measure interactions between endogenous Exosome Complex components in erythroid cells. This assay is being used to establish the role of the various Exosome Complex subunits in Complex integrity in cultured and primary erythroid cells. Under conditions in which downregulating Exosc8 or Exosc9 induced erythroid maturation, expression of the established regulators of erythropoiesis GATA-1, FOG-1, or KLF1 was unaffected. To test the hypothesis that the Exosome Complex downregulates a cohort of critical RNAs, including regulatory non-coding RNAs, required for erythroid maturation, we are conducting studies to identify direct Exosome Complex targets in erythroid cells at distinct stages of maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the Exosome Complex are endogenous suppressors of the erythroid developmental program. Furthermore, since the Exosome Complex had not been shown previously to regulate any aspect of hematopoiesis, this work expands the biological repertoire of Exosome Complex-dependent processes. Disclosures No relevant conflicts of interest to declare.