RESUMEN
Ribosome synthesis begins in the nucleolus with 90S pre-ribosome construction, but little is known about how the many different snoRNAs that modify the pre-rRNA are timely guided to their target sites. Here, we report a role for Cms1 in such a process. Initially, we discovered CMS1 as a null suppressor of a nop14 mutant impaired in Rrp12-Enp1 factor recruitment to the 90S. Further investigations detected Cms1 at the 18S rRNA 3' major domain of an early 90S that carried H/ACA snR83, which is known to guide pseudouridylation at two target sites within the same subdomain. Cms1 co-precipitates with many 90S factors, but Rrp12-Enp1 encircling the 3' major domain in the mature 90S is decreased. We suggest that Cms1 associates with the 3' major domain during early 90S biogenesis to restrict premature Rrp12-Enp1 binding but allows snR83 to timely perform its modification role before the next 90S assembly steps coupled with Cms1 release take place.
Asunto(s)
Nucléolo Celular , Ribosomas , Ribosomas/metabolismo , Nucléolo Celular/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Nucleolar Pequeño/metabolismoRESUMEN
Site-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal "antisense" sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5'-UN-3', including the 5' substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (Ψ) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs.
Asunto(s)
Seudouridina , ARN Guía de Kinetoplastida , Humanos , Conformación de Ácido Nucleico , Seudouridina/química , ARN/química , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , ARN Ribosómico , UridinaRESUMEN
Cytoplasmic localization, stability, and translation of mRNAs are controlled by their dynamic association of numerous mRNA-binding (mRNP) proteins, including cold shock domain (CSD)-containing proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and serine/arginine-rich (SR) proteins. Here, we demonstrate that the most abundant human mRNP protein, the CSD-containing Y-box-binding protein 1 (YBX1), the closely related YBX3 protein, and other mRNP proteins, such as SRSF1, SRSF2, SRSF3, hnRNP A1, and H, specifically and efficiently interact with overlapping sets of mitochondrial tRNAs (mt tRNAs). In vitro reconstitution and in vivo binding experiments show that YBX1 recognizes the D- and/or T-stem-loop regions of mt tRNAs through relying on the RNA-binding capacity of its CSD. Cell fractionation and in vivo RNA-protein cross-linking experiments demonstrate that YBX1 and YBX3 interact with mt tRNAs in the cytosol outside of mitochondria. Cell fractionation and fluorescence in situ hybridization experiments provide evidence that mitochondrial autophagy promotes the release of mt tRNAs from the mitochondria into the cytoplasm. Association of mRNP proteins with mt tRNAs is highly dynamic; it is rapidly increased upon transcription inhibition and decreased during apoptosis. Although the cytoplasmic function of mt tRNAs remains elusive, their dynamic interactions with key mRNA-binding proteins may influence cytoplasmic mRNA stability and/or translation.
Asunto(s)
Citosol/química , Mitocondrias/química , ARN de Transferencia/química , Ribonucleoproteínas/química , Autofagia/genética , Ribonucleoproteína Nuclear Heterogénea A1/química , Ribonucleoproteína Nuclear Heterogénea A1/genética , Humanos , Hibridación Fluorescente in Situ , Mitocondrias/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN de Transferencia/genética , Ribonucleoproteínas/genética , Factores de Empalme Serina-Arginina/química , Factores de Empalme Serina-Arginina/genética , Proteína 1 de Unión a la Caja Y/química , Proteína 1 de Unión a la Caja Y/genéticaRESUMEN
Cytoplasmic maturation of precursors to the small ribosomal subunit in yeast requires the intervention of a dozen assembly factors (AFs), the precise roles of which remain elusive. One of these is Rio1p that seems to intervene at a late step of pre-40S particle maturation. We have investigated the role played by Rio1p in the dynamic association and dissociation of AFs with and from pre-40S particles. Our results indicate that Rio1p depletion leads to the stalling of at least 4 AFs (Nob1p, Tsr1p, Pno1p/Dim2p and Fap7p) in 80S-like particles. We conclude that Rio1p is important for the timely release of these factors from 80S-like particles. In addition, we present immunoprecipitation and electron microscopy evidence suggesting that when Rio1p is depleted, a subset of Nob1p-containing pre-40S particles associate with translating polysomes. Using Nob1p as bait, we purified pre-40S particles from cells lacking Rio1p and performed ribosome profiling experiments which suggest that immature 40S subunits can carry out translation elongation. We conclude that lack of Rio1p allows premature entry of pre-40S particles in the translation process and that the presence of Nob1p and of the 18S rRNA 3' extension in the 20S pre-rRNA is not incompatible with translation elongation.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/fisiología , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas Nucleares/metabolismo , Extensión de la Cadena Peptídica de Translación , Polirribosomas/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Mammalian cells express hundreds of intron-encoded box H/ACA RNAs which fold into a common hairpin-hinge-hairpin-tail structure, interact with 4 evolutionarily conserved proteins, dyskerin, Nop10, Nhp2 and Gar1, and function mainly in RNA pseudouridylation. The human telomerase H/ACA RNA (hTR) directs telomeric DNA synthesis and it carries a 5'-terminal domain encompassing the telomeric template sequence. The primary hTR transcript is synthesized from an independent gene by RNA polymerase II and undergoes 3' end processing controlled by the 3'-terminal H/ACA domain. The apical stem-loop of the 3' hairpin of hTR carries a unique biogenesis-promoting element, the BIO motif that promotes hTR processing and RNP assembly. AluACA RNAs represent a distinct class of human H/ACA RNAs; they are processed from intronic Alu repetitive sequences. As compared to canonical H/ACA RNAs, the AluACA RNAs carry unusually short or long 5' hairpins and generally, they accumulate at low levels. Here, we demonstrate that the suboptimal 5' hairpins are responsible for the weak expression of AluACA RNAs. We also show that AluACA RNAs frequently carry a processing/stabilization element that is structurally and functionally indistinguishable from the hTR BIO motif. Both hTR and AluACA biogenesis-promoting elements are located in the terminal stem-loop of the 3'-terminal H/ACA hairpin, they show perfect structural conservation and are functionally interchangeable in in vivo RNA processing reactions. Our results demonstrate that the BIO motif, instead of being confined to hTR, is a more general H/ACA RNP biogenesis-facilitating element that can also promote processing/assembly of intron-encoded AluACA RNPs.
Asunto(s)
ARN Polimerasa II/metabolismo , ARN/química , ARN/metabolismo , Expresión Génica , Células HeLa , Humanos , Intrones , Modelos Moleculares , Conformación de Ácido Nucleico , Edición de ARN , Telomerasa/química , Telomerasa/metabolismo , Telómero/genéticaRESUMEN
Human polypyrimidine tract-binding protein PTB is a multifunctional RNA-binding protein with four RNA recognition motifs (RRM1 to RRM4). PTB is a nucleocytoplasmic shuttle protein that functions as a key regulator of alternative pre-mRNA splicing in the nucleoplasm and promotes internal ribosome entry site-mediated translation initiation of viral and cellular mRNAs in the cytoplasm. Here, we demonstrate that PTB and its paralogs, nPTB and ROD1, specifically interact with mitochondrial (mt) tRNA(Thr) both in human and mouse cells. In vivo and in vitro RNA-binding experiments demonstrate that PTB forms a direct interaction with the T-loop and the D-stem-loop of mt tRNA(Thr) using its N-terminal RRM1 and RRM2 motifs. RNA sequencing and cell fractionation experiments show that PTB associates with correctly processed and internally modified, mature mt tRNA(Thr) in the cytoplasm outside of mitochondria. Consistent with this, PTB activity is not required for mt tRNA(Thr) biogenesis or for correct mitochondrial protein synthesis. PTB association with mt tRNA(Thr) is largely increased upon induction of apoptosis, arguing for a potential role of the mt tRNA(Thr)/PTB complex in apoptosis. Our results lend strong support to the recently emerging conception that human mt tRNAs can participate in novel cytoplasmic processes independent from mitochondrial protein synthesis.
Asunto(s)
Citoplasma/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , ARN de Transferencia de Treonina/metabolismo , Secuencias de Aminoácidos/genética , Animales , Apoptosis/genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Células HEK293 , Células HeLa , Humanos , Ratones , Mitocondrias/genética , Datos de Secuencia Molecular , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Conformación de Ácido Nucleico , Proteína de Unión al Tracto de Polipirimidina/genética , Unión Proteica , Interferencia de ARN , ARN de Transferencia de Treonina/química , ARN de Transferencia de Treonina/genéticaRESUMEN
Alu repetitive sequences are the most abundant short interspersed DNA elements in the human genome. Full-length Alu elements are composed of two tandem sequence monomers, the left and right Alu arms, both derived from the 7SL signal recognition particle RNA. Since Alu elements are common in protein-coding genes, they are frequently transcribed into pre-mRNAs. Here, we demonstrate that the right arms of nascent Alu transcripts synthesized within pre-mRNA introns are processed into metabolically stable small RNAs. The intron-encoded Alu RNAs, termed AluACA RNAs, are structurally highly reminiscent of box H/ACA small Cajal body (CB) RNAs (scaRNAs). They are composed of two hairpin units followed by the essential H (AnAnnA) and ACA box motifs. The mature AluACA RNAs associate with the four H/ACA core proteins: dyskerin, Nop10, Nhp2, and Gar1. Moreover, the 3' hairpin of AluACA RNAs carries two closely spaced CB localization motifs, CAB boxes (UGAG), which bind Wdr79 in a cumulative fashion. In contrast to canonical H/ACA scaRNPs, which concentrate in CBs, the AluACA RNPs accumulate in the nucleoplasm. Identification of 348 human AluACA RNAs demonstrates that intron-encoded AluACA RNAs represent a novel, large subgroup of H/ACA RNAs, which are apparently confined to human or primate cells.
Asunto(s)
Elementos Alu/fisiología , Intrones , Proteínas/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Expresión Génica , Células HeLa , Humanos , Chaperonas Moleculares , Estructura Secundaria de Proteína , ARN/química , ARN/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/química , Telomerasa , Globinas beta/genéticaRESUMEN
Yeast snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) that promotes 18S rRNA processing through forming transient base-pairing interactions with the newly synthesized 35S pre-rRNA. By using a novel tandem RNA affinity selection approach, followed by coimmunoprecipitation and in vivo cross-linking experiments, we demonstrate that in addition to the four H/ACA core proteins, Cbf5p, Nhp2p, Nop10p and Gar1p, a fraction of snR30 specifically associates with the Utp23p and Kri1p nucleolar proteins. Depletion of Utp23p and Kri1p has no effect on the accumulation and recruitment of snR30 to the nascent pre-ribosomes. However, in the absence of Utp23p, the majority of snR30 accumulates in large pre-ribosomal particles. The retained snR30 is not base-paired with the 35S pre-rRNA, indicating that its aberrant tethering to nascent preribosomes is likely mediated by pre-ribosomal protein(s). Thus, Utp23p may promote conformational changes of the pre-ribosome, essential for snR30 release. Neither Utp23p nor Kri1p is required for recruitment of snR30 to the nascent pre-ribosome. On the contrary, depletion of snR30 prevents proper incorporation of both Utp23p and Kri1p into the 90S pre-ribosome containing the 35S pre-rRNA, indicating that snR30 plays a central role in the assembly of functionally active small subunit processome.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN Nucleolar Pequeño/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/aislamiento & purificación , Ribonucleoproteínas Nucleolares Pequeñas/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.
Asunto(s)
VIH-1/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/fisiología , Región de Flanqueo 5'/genética , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , Regulación Viral de la Expresión Génica , VIH-1/genética , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Multimerización de Proteína , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción , Activación Transcripcional/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Box H/ACA RNAs represent an abundant, evolutionarily conserved class of small noncoding RNAs. All H/ACA RNAs associate with a common set of proteins, and they function as ribonucleoprotein (RNP) enzymes mainly in the site-specific pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Some H/ACA RNPs function in the nucleolytic processing of precursor rRNA (pre-rRNA) and synthesis of telomeric DNA. Thus, H/ACA RNPs are essential for three fundamental cellular processes: protein synthesis, mRNA splicing, and maintenance of genome integrity. Recently, great progress has been made toward understanding of the biogenesis, intracellular trafficking, structure, and function of H/ACA RNPs.
Asunto(s)
Cuerpos Enrollados/metabolismo , ARN no Traducido/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proteínas Arqueales/metabolismo , Evolución Molecular , Inestabilidad Genómica , Humanos , Transferasas Intramoleculares/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Conformación Proteica , Seudouridina/metabolismo , Procesamiento Postranscripcional del ARN , Empalme del ARN , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , ARN no Traducido/química , Ribonucleoproteínas Nucleares Pequeñas/biosíntesis , Ribonucleoproteínas Nucleares Pequeñas/química , Telomerasa/metabolismoRESUMEN
The U1 small nuclear RNA (snRNA)--in the form of the U1 spliceosomal Sm small nuclear ribonucleoprotein particle (snRNP) that contains seven Sm and three U1-specific RNP proteins-has a crucial function in the recognition and removal of pre-messenger RNA introns. Here, we show that a fraction of human U1 snRNA specifically associates with the nuclear RNA-binding protein TBP-associated factor 15 (TAF15). We show that none of the known protein components of the spliceosomal U1-Sm snRNP interacts with the newly identified U1-TAF15 snRNP. In addition, the U1-TAF15 snRNP tightly associates with chromatin in an RNA-dependent manner and accumulates in nucleolar caps upon transcriptional inhibition. The Sm-binding motif of U1 snRNA is essential for the biogenesis of both U1-Sm and U1-TAF15 snRNPs, suggesting that the U1-TAF15 particle is produced by remodelling of the U1-Sm snRNP. A demonstration that human U1 snRNA forms at least two structurally distinct snRNPs supports the idea that the U1 snRNA has many nuclear functions.
Asunto(s)
ARN Nuclear Pequeño/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Secuencia de Bases , Western Blotting , Cromatina/genética , Cromatina/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Inmunoprecipitación , Hibridación in Situ , Unión Proteica , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genéticaRESUMEN
RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo L/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Secuencia Conservada/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Proteínas HSP90 de Choque Térmico/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo L/genética , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica/fisiología , Pliegue de Proteína , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/fisiología , Factores de TranscripciónRESUMEN
The RNA component of human telomerase (hTR) includes H/ACA and CR7 domains required for 3' end processing, localization, and accumulation. The terminal loop of the CR7 domain contains the CAB box (ugAG) required for targeting of scaRNAs to Cajal bodies (CB) and an uncharacterized sequence required for accumulation and processing. To dissect out the contributions of the CR7 stem loop to hTR processing and localization, we solved the solution structures of the 3' terminal stem loops of hTR CR7 and U64 H/ACA snoRNA, and the 5' terminal stem loop of U85 C/D-H/ACA scaRNA. These structures, together with analysis of localization, processing, and accumulation of hTRs containing nucleotide substitutions in the CR7 domain, identified the sequence and structural requirements of the hTR processing and CB localization signals and showed that these signals are functionally independent. Further, 3' end processing was found to be a prerequisite for translocation of hTR to CBs.
Asunto(s)
Cuerpos Enrollados/metabolismo , Procesamiento de Término de ARN 3' , Transporte de ARN , ARN/química , ARN/metabolismo , Telomerasa/química , Telomerasa/metabolismo , Emparejamiento Base , Secuencia de Bases , Cisteína/genética , Disqueratosis Congénita/genética , Glicina/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación/genética , Nucleótidos/química , ARN/genética , ARN Nucleolar Pequeño/química , Soluciones , Telomerasa/genéticaRESUMEN
The 7SK small nuclear RNA (snRNA) regulates RNA polymerase II transcription elongation by controlling the protein kinase activity of the positive transcription elongation factor b (P-TEFb). In cooperation with HEXIM1, the 7SK snRNA sequesters P-TEFb into the kinase-inactive 7SK/HEXIM1/P-TEFb small nuclear ribonucleoprotein (snRNP), and thereby, controls the nuclear level of active P-TEFb. Here, we report that a fraction of HeLa 7SK snRNA that is not involved in 7SK/HEXIM1/P-TEFb formation, specifically interacts with RNA helicase A (RHA), heterogeneous nuclear ribonucleoprotein A1 (hnRNP), A2/B1, R and Q proteins. Inhibition of cellular transcription induces disassembly of 7SK/HEXIM1/P-TEFb and at the same time, increases the level of 7SK snRNPs containing RHA, hnRNP A1, A2/B1, R and Q. Removal of transcription inhibitors restores the original levels of the 7SK/HEXIM1/P-TEFb and '7SK/hnRNP' complexes. 7SK/HEXIM1/P-TEFb snRNPs containing mutant 7SK RNAs lacking the capacity for binding hnRNP A1, A2, R and Q are resistant to stress-induced disassembly, indicating that recruitment of the novel 7SK snRNP proteins is essential for disruption of 7SK/HEXIM1/P-TEFb. Thus, we propose that the nuclear level of active P-TEFb is controlled by dynamic and reversible remodelling of 7SK snRNP.
Asunto(s)
Núcleo Celular/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Secuencia de Bases , Cromatografía de Afinidad , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , ARN/química , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/aislamiento & purificaciónRESUMEN
Telomerase is a ribonucleoprotein enzyme that counteracts replicative telomere erosion by adding telomeric sequence repeats onto chromosome ends. Despite its well-established role in telomere synthesis, telomerase has not yet been detected at telomeres. The RNA component of human telomerase (hTR) resides in the nucleoplasmic Cajal bodies (CBs) of interphase cancer cells. Here, in situ hybridization demonstrates that in human HeLa and Hep2 S phase cells, besides accumulating in CBs, hTR specifically concentrates at a few telomeres that also accumulate the TRF1 and TRF2 telomere marker proteins. Surprisingly, telomeres accumulating hTR exhibit a great accessibility for in situ oligonucleotide hybridization without chromatin denaturation, suggesting that they represent a structurally distinct, minor subset of HeLa telomeres. Moreover, we demonstrate that more than 25% of telomeres accumulating hTR colocalize with CBs. Time-lapse fluorescence microscopy demonstrates that CBs moving in the nucleoplasm of S phase cells transiently associate for 10-40 min with telomeres. Our data raise the intriguing possibility that CBs may deliver hTR to telomeres and/or may function in other aspects of telomere maintenance.
Asunto(s)
Cuerpos Enrollados/metabolismo , ARN no Traducido/análisis , Telomerasa/análisis , Telómero/metabolismo , Ciclo Celular/fisiología , Núcleo Celular/ultraestructura , Cuerpos Enrollados/ultraestructura , Células HeLa , Humanos , Hibridación Fluorescente in Situ , ARN , ARN Largo no Codificante , ARN no Traducido/metabolismo , Proteínas Recombinantes de Fusión/análisis , Fase S/fisiología , Telomerasa/metabolismo , Telómero/ultraestructuraRESUMEN
To better understand intranuclear-targeting mechanisms, we have studied the transport of U3 snoRNA in human cells. Surprisingly, we found that PHAX, the snRNA export adaptor, is highly enriched in complexes containing m7G-capped U3 precursors. In contrast, the export receptor CRM1 is predominantly bound to TMG-capped U3 species. In agreement, PHAX does not export m7G-capped U3 precursors because their caps become hypermethylated in the nucleus. Inactivation of PHAX and CRM1 shows that U3 first requires PHAX to reach Cajal bodies, and then CRM1 to be routed from there to nucleoli. Furthermore, PHAX also binds the precursors of U8 and U13 box C/D snoRNAs and telomerase RNA. PHAX was previously shown to discriminate between small versus large RNAs during export. Our data indicate that the role of PHAX in determining the identity of small RNAs extends to nonexported species, and this appears critical to promote their transport within the nucleus.
Asunto(s)
Nucléolo Celular/metabolismo , Carioferinas/fisiología , Proteínas de Transporte Nucleocitoplasmático/fisiología , Fosfoproteínas/fisiología , ARN Nucleolar Pequeño/química , Receptores Citoplasmáticos y Nucleares/fisiología , Secuencias de Aminoácidos , Transporte Biológico , Técnicas de Cultivo de Célula , Línea Celular , Núcleo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Metilación de ADN , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Hibridación in Situ , Microscopía Fluorescente , Plásmidos/metabolismo , ARN/química , ARN/metabolismo , ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Telomerasa/metabolismo , Transfección , Proteína Exportina 1RESUMEN
Pseudouridine, the most abundant modified nucleoside in RNA, is synthesized by posttranscriptional isomerization of uridines. In eukaryotic RNAs, site-specific synthesis of pseudouridines is directed primarily by box H/ACA guide RNAs. In this study, we have identified 61 novel putative pseudouridylation guide RNAs by construction and characterization of a cDNA library of human box H/ACA RNAs. The majority of the new box H/ACA RNAs are predicted to direct pseudouridine synthesis in rRNAs and spliceosomal small nuclear RNAs. We can attribute RNA-directed modification to 79 of the 97 pseudouridylation sites present in the human 18S, 5.8S, and 28S rRNAs and to 11 of the 21 pseudouridines reported for the U1, U2, U4, U5, and U6 spliceosomal RNAs. We have also identified 12 novel box H/ACA RNAs which lack apparent target pseudouridines in rRNAs and small nuclear RNAs. These putative guide RNAs likely function in the pseudouridylation of some other types of cellular RNAs, suggesting that RNA-guided pseudouridylation is more general than assumed before. The genomic organization of the new box H/ACA RNA genes indicates that in human cells, all box H/ACA pseudouridylation guide RNAs are processed from introns of pre-mRNA transcripts which either encode a protein product or lack protein-coding capacity.
Asunto(s)
Seudouridina/biosíntesis , Procesamiento Postranscripcional del ARN , Biblioteca de Genes , Genes de ARNr , Genoma Humano , Humanos , Intrones , Sustancias Macromoleculares , Precursores del ARN/metabolismo , ARN Ribosómico/biosíntesis , ARN Pequeño no TraducidoRESUMEN
Pseudouridines and 2'-O-methylated nucleotides are ubiquitous constituents of stable cellular RNAs. In eukaryotes, posttranscriptional synthesis of most pseudouridines and 2'-O-methylated nucleotides is directed by sequence-specific guide RNAs (gRNAs). In recent years, an enormous number of novel putative modification gRNAs have been identified in a broad variety of organisms by using bioinformatics and large-scale cDNA-sequencing approaches. Understanding of the function of the novel modification gRNAs as well as the functional importance of modified nucleotides requires techniques that support the site-specific detection of 2'-O-methylated nucleotides and pseudouridines. Here, we describe rapid, reverse transcription-based methods to map 2'-O-methylated nucleotides and pseudouridines in any cellular RNA.
Asunto(s)
Seudouridina/análisis , Ribonucleótidos/aislamiento & purificación , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida/métodos , Metilación , Oligodesoxirribonucleótidos/aislamiento & purificación , Procesamiento Postranscripcional del ARN/genética , Ribonucleótidos/química , ARNt Metiltransferasas , ARN Pequeño no TraducidoRESUMEN
Telomerase is a ribonucleoprotein reverse transcriptase that uses its RNA component as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes. Here, fluorescence in situ hybridization demonstrates that in HeLa cancer cells, human telomerase RNA (hTR) accumulates in the nucleoplasmic Cajal bodies (CBs). Localization of transiently expressed hTR to CBs is supported by a short sequence motif (411-UGAG-414) that is located in the 3'-terminal box H/ACA RNA-like domain of hTR and that is structurally and functionally indistinguishable from the CB-specific localization signal of box H/ACA small CB-specific RNAs. In synchronized HeLa cells, hTR shows the most efficient accumulation in CBs during S phase, when telomeres are most likely synthesized. CBs may function in post-transcriptional maturation (e.g., cap hypermethylation of hTR), but they may also play a role in the assembly and/or function of telomerase holoenzyme.
Asunto(s)
Cuerpos Enrollados/metabolismo , Señales de Clasificación de Proteína , ARN Nucleolar Pequeño , ARN/metabolismo , Telomerasa/metabolismo , Secuencias de Aminoácidos , Ciclo Celular/fisiología , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Conformación de Ácido Nucleico , ARN/química , ARN/genética , Caperuzas de ARN , Telomerasa/química , Telomerasa/genéticaRESUMEN
Post-transcriptional synthesis of 2'-O-methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modification in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and specifically targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D-H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body-specific localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5' and 3' hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism.