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1.
Nucleic Acids Res ; 52(4): 1953-1974, 2024 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-38113271

RESUMEN

Regulation of RNA helicase activity, often accomplished by protein cofactors, is essential to ensure target specificity within the complex cellular environment. The largest family of RNA helicase cofactors are the G-patch proteins, but the cognate RNA helicases and cellular functions of numerous human G-patch proteins remain elusive. Here, we discover that GPATCH4 is a stimulatory cofactor of DHX15 that interacts with the DEAH box helicase in the nucleolus via residues in its G-patch domain. We reveal that GPATCH4 associates with pre-ribosomal particles, and crosslinks to the transcribed ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated RNAs that guide rRNA and snRNA modifications. Loss of GPATCH4 impairs 2'-O-methylation at various rRNA and snRNA sites leading to decreased protein synthesis and cell growth. We demonstrate that the regulation of 2'-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2'-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings extend knowledge on RNA helicase regulation by G-patch proteins and also provide important new insights into the mechanisms regulating installation of rRNA and snRNA modifications, which are essential for ribosome function and pre-mRNA splicing.


Asunto(s)
ARN Helicasas , ARN Ribosómico , Humanos , Metilación , Ribosomas/metabolismo , ARN Helicasas/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo
2.
Nucleic Acids Res ; 49(7): 4066-4084, 2021 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-33721027

RESUMEN

RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2'-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and 'free' pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2'-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Escherichia coli , Metilación , Precursores del ARN/metabolismo , Levaduras/enzimología
3.
Nucleic Acids Res ; 48(1): 405-420, 2020 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-31745560

RESUMEN

More than 200 assembly factors (AFs) are required for the production of ribosomes in yeast. The stepwise association and dissociation of these AFs with the pre-ribosomal subunits occurs in a hierarchical manner to ensure correct maturation of the pre-rRNAs and assembly of the ribosomal proteins. Although decades of research have provided a wealth of insights into the functions of many AFs, others remain poorly characterized. Pol5 was initially classified with B-type DNA polymerases, however, several lines of evidence indicate the involvement of this protein in ribosome assembly. Here, we show that depletion of Pol5 affects the processing of pre-rRNAs destined for the both the large and small subunits. Furthermore, we identify binding sites for Pol5 in the 5' external transcribed spacer and within domain III of the 25S rRNA sequence. Consistent with this, we reveal that Pol5 is required for recruitment of ribosomal proteins that form the polypeptide exit tunnel in the LSU and that depletion of Pol5 impairs the release of 5' ETS fragments from early pre-40S particles. The dual functions of Pol5 in 60S assembly and recycling of pre-40S AFs suggest that this factor could contribute to ensuring the stoichiometric production of ribosomal subunits.


Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Biosíntesis de Proteínas , ARN Ribosómico/genética , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sitios de Unión , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Termodinámica
4.
RNA Biol ; 18(8): 1124-1135, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33048000

RESUMEN

RNA helicases contribute to diverse aspects of RNA metabolism through their functions in re-arranging RNA structures. Identification of the remodelling targets of RNA helicases is a critical step in elucidating their cellular functions. Here, we show that, in contrast to many other ribosome biogenesis factors, the DExD box ATPase DDX55 predominantly localizes to the nucleoplasm and we identify a nuclear localization signal within the C-terminal region of the protein. DDX55 associates with pre-ribosomal subunits in human cells and is required for maturation of large subunit pre-rRNAs. Interestingly, in vitro analyses show that DDX55 selectively associates with double-stranded RNA substrates, which also stimulate its ATPase activity, and our data suggest that the C-terminal region of DDX55 contributes to this substrate specificity. The C-terminal region of DDX55 is also necessary for recruitment of the helicase to pre-ribosomes and, using in vivo crosslinking, we reveal a binding site for DDX55 in helix H62 of the 28S ribosomal RNA. Taken together, these data highlight the importance of the C-terminal region of DDX55 in substrate specificity and recruitment, and identify domain IV as a potential remodelling target of DDX55 during LSU biogenesis.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico 28S/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , Células HEK293 , Células HeLa , Humanos , MicroARNs/química , MicroARNs/genética , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Biogénesis de Organelos , Unión Proteica , Biosíntesis de Proteínas , Dominios y Motivos de Interacción de Proteínas , Precursores del ARN/química , Precursores del ARN/genética , ARN Largo no Codificante/química , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Ribosómico 28S/química , ARN Ribosómico 28S/genética , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribosomas/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido
5.
Nucleic Acids Res ; 47(15): 7719-7733, 2019 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-31328227

RESUMEN

N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5-TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5-TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.


Asunto(s)
Adenosina/química , Regulación Neoplásica de la Expresión Génica , Metiltransferasas/química , ARN Mensajero/química , ARN Ribosómico 18S/química , Adenosina/genética , Adenosina/metabolismo , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Cristalografía por Rayos X , Eliminación de Gen , Células HCT116 , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Transducción de Señal , Especificidad por Sustrato
6.
RNA ; 24(10): 1339-1350, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29970596

RESUMEN

N6-methyladenosine (m6A) modifications in RNAs play important roles in regulating many different aspects of gene expression. While m6As can have direct effects on the structure, maturation, or translation of mRNAs, such modifications can also influence the fate of RNAs via proteins termed "readers" that specifically recognize and bind modified nucleotides. Several YTH domain-containing proteins have been identified as m6A readers that regulate the splicing, translation, or stability of specific mRNAs. In contrast to the other YTH domain-containing proteins, YTHDC2 has several defined domains and here, we have analyzed the contribution of these domains to the RNA and protein interactions of YTHDC2. The YTH domain of YTHDC2 preferentially binds m6A-containing RNAs via a conserved hydrophobic pocket, whereas the ankyrin repeats mediate an RNA-independent interaction with the 5'-3' exoribonuclease XRN1. We show that the YTH and R3H domains contribute to the binding of YTHDC2 to cellular RNAs, and using crosslinking and analysis of cDNA (CRAC), we reveal that YTHDC2 interacts with the small ribosomal subunit in close proximity to the mRNA entry/exit sites. YTHDC2 was recently found to promote a "fast-track" expression program for specific mRNAs, and our data suggest that YTHDC2 accomplishes this by recruitment of the RNA degradation machinery to regulate the stability of m6A-containing mRNAs and by utilizing its distinct RNA-binding domains to bridge interactions between m6A-containing mRNAs and the ribosomes to facilitate their efficient translation.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina/análogos & derivados , Exorribonucleasas/metabolismo , Subunidades Ribosómicas Pequeñas/metabolismo , Adenosina/química , Adenosina/metabolismo , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN/química , ARN/genética , ARN/metabolismo , ARN Helicasas , Relación Estructura-Actividad
7.
RNA Biol ; 17(4): 539-553, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31994962

RESUMEN

Assembly of eukaryotic ribosomal subunits is a complex and dynamic process involving the action of more than 200 trans-acting assembly factors. Although recent cryo-electron microscopy structures have provided information on architecture of several pre-ribosomal particles and the binding sites of many AFs, the RNA and protein interactions of many other AFs not captured in these snapshots still remain elusive. RNA helicases are key regulators of structural rearrangements within pre-ribosomal complexes and here we have analysed the eIF4A-like RNA helicase Fal1 and its putative cofactor Sgd1. Our data show that these proteins interact directly via the MIF4G domain of Sgd1 and that the MIF4G domain of Sgd1 stimulates the catalytic activity of Fal1 in vitro. The catalytic activity of Fal1, and the interaction between Fal1 and Sgd1, are required for efficient pre-rRNA processing at the A0, A1 and A2 sites. Furthermore, Sgd1 co-purifies the early small subunit biogenesis factors Lcp5 and Rok1, suggesting that the Fal1-Sgd1 complex likely functions within the SSU processome. In vivo crosslinking data reveal that Sgd1 binds to helix H12 of the 18S rRNA sequence and we further demonstrate that this interaction is formed by the C-terminal region of the protein, which is essential for its function in ribosome biogenesis.


Asunto(s)
Proteínas Nucleares/metabolismo , ARN Ribosómico 18S/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Modelos Moleculares , Proteínas Nucleares/química , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Dominios Proteicos , ARN Ribosómico 18S/química , Proteínas de Unión al ARN/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química
8.
EMBO Rep ; 18(11): 2004-2014, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-29051200

RESUMEN

N6-methyladenosine (m6A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m6A modifications are installed by the METTL3-METTL14 complex, others appear to be introduced independently, implying that additional human m6A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m6A "writer" protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre-mRNAs. We demonstrate that METTL16 is responsible for N6-methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5' splice sites of pre-mRNAs during splicing, suggesting that METTL16-mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m6A methyltransferase in human cells expands our understanding of the mechanisms by which the m6A landscape is installed on cellular RNAs.


Asunto(s)
Adenosina/análogos & derivados , Metiltransferasas/genética , Precursores del ARN/genética , Empalme del ARN , ARN Largo no Codificante/metabolismo , ARN Nuclear Pequeño/metabolismo , Adenosina/metabolismo , Emparejamiento Base , Secuencia de Bases , ADN Complementario/genética , ADN Complementario/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Metilación , Metiltransferasas/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Precursores del ARN/metabolismo , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
9.
RNA Biol ; 16(1): 54-68, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30582406

RESUMEN

Ribosome synthesis is an essential cellular process, and perturbation of human ribosome production is linked to cancer and genetic diseases termed ribosomopathies. During their assembly, pre-ribosomal particles undergo numerous structural rearrangements, which establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome biogenesis. RNA helicases are essential mediators of such remodelling events and here, we demonstrate that the DEAH-box RNA helicase DHX37 is required for maturation of the small ribosomal subunit in human cells. Our data reveal that the presence of DHX37 in early pre-ribosomal particles is monitored by a quality control pathway and that failure to recruit DHX37 leads to pre-rRNA degradation. Using an in vivo crosslinking approach, we show that DHX37 binds directly to the U3 small nucleolar RNA (snoRNA) and demonstrate that the catalytic activity of the helicase is required for dissociation of the U3 snoRNA from pre-ribosomal complexes. This is an important event during ribosome assembly as it enables formation of the central pseudoknot structure of the small ribosomal subunit. We identify UTP14A as a direct interaction partner of DHX37 and our data suggest that UTP14A can act as a cofactor that stimulates the activity of the helicase in the context of U3 snoRNA release.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Ribosomas/metabolismo , Catálisis , Línea Celular Tumoral , Humanos , Unión Proteica , Procesamiento Postranscripcional del ARN , ARN Nucleolar Pequeño/genética
10.
RNA ; 20(8): 1173-82, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24947498

RESUMEN

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the "foot" region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Emparejamiento Base , Conformación de Ácido Nucleico , Unión Proteica , Precursores del ARN/química , Precursores del ARN/genética , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
11.
RNA Biol ; 13(3): 320-30, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26821976

RESUMEN

A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Apoptosis , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación Fúngica de la Expresión Génica , Membranas Mitocondriales/metabolismo , Transducción de Señal
12.
Nat Commun ; 13(1): 209, 2022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-35017528

RESUMEN

Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m3C32 in the human mitochondrial (mt-)tRNAThr and mt-tRNASer(UCN). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed U34G35 and t6A37/(ms2)i6A37, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C32. Several lines of evidence demonstrate the influence of U34, G35, and the m3C32 and t6A37/(ms2)i6A37 modifications in mt-tRNAThr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNAThr/Ser(UCN) lacking METTL8-mediated m3C32 are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m3C32 within mt-tRNAs.


Asunto(s)
Anticodón/química , Metiltransferasas/genética , Mitocondrias/genética , ARN Mitocondrial/química , ARN de Transferencia de Serina/química , ARN de Transferencia de Treonina/química , Anticodón/metabolismo , Emparejamiento Base , Citosina/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Metilación , Metiltransferasas/metabolismo , Mitocondrias/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Biosíntesis de Proteínas , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN de Transferencia de Serina/genética , ARN de Transferencia de Serina/metabolismo , ARN de Transferencia de Treonina/genética , ARN de Transferencia de Treonina/metabolismo , Transducción de Señal
13.
Nat Commun ; 12(1): 782, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542241

RESUMEN

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatasas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Mutación , Terminación de la Cadena Péptídica Traduccional , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Partícula de Reconocimiento de Señal/metabolismo , Ubiquitina/genética , Ubiquitina/aislamiento & purificación
14.
Nat Commun ; 12(1): 6152, 2021 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-34686661

RESUMEN

Early pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre-60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center (PTC) and is responsible for stabilizing interactions between the 5' and 3' ends of the 25S, an essential pre-requisite for subsequent pre-60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , ARN Ribosómico/química , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , ARN Helicasas DEAD-box/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Pliegue del ARN , Precursores del ARN/química , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/metabolismo , Proteína Ribosomal L3/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
15.
Nat Commun ; 11(1): 5535, 2020 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-33139697

RESUMEN

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.


Asunto(s)
ADN Helicasas/genética , Reparación del ADN , Neoplasias/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Secuencia Conservada/genética , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Células HEK293 , Humanos , Mutación , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Unión Proteica/genética , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/metabolismo
16.
Nat Commun ; 9(1): 5383, 2018 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-30568249

RESUMEN

Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly. Using in vivo crosslinking, we here identify the pre-ribosomal binding sites of three RNA helicases. Our data support roles for Has1 in triggering release of the U14 snoRNP, a critical event during early 40S maturation, and in driving assembly of domain I of pre-60S complexes. Binding of Mak5 to domain II of pre-60S complexes promotes recruitment of the ribosomal protein Rpl10, which is necessary for subunit joining and ribosome function. Spb4 binds to a molecular hinge at the base of ES27 facilitating binding of the export factor Arx1, thereby promoting pre-60S export competence. Our data provide important insights into the driving forces behind key structural remodelling events during ribosomal subunit assembly.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Subunidades Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae
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