The nuclear SMN complex localizes to specific structures called nuclear gems. The loss of gems is a cellular marker for several neurodegenerative diseases. Here, we identify that the U1-snRNP-specific protein U1-70K localizes to nuclear gems, and we show that U1-70K is necessary for gem integrity. Furthermore, we show that the interaction between U1-70K and the SMN complex is RNA independent, and we map the SMN complex binding site to the unstructured N-terminal tail of U1-70K. Consistent with these results, the expression of the U1-70K N-terminal tail rescues gem formation. These findings show that U1-70K is an SMN-complex-associating protein, and they suggest a new function for U1-70K in the formation of nuclear gems.

• Klíčová slova
• Cajal bodies, Gems, Nuclear structure, SMN, U1 snRNP, U1-70K,
• MeSH
• asociované struktury Cajalových tělísek chemie   metabolismus   MeSH
• buněčné jádro chemie   genetika   metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U1 chemie   genetika   metabolismus   MeSH
• proteinový komplex SMN genetika   metabolismus   MeSH
• sestřih RNA MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U1 MeSH
• proteinový komplex SMN MeSH
• SNRNP70 protein, human MeSH Prohlížeč

Splicing in S. cerevisiae has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the AMA1 intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in prp45(1-169) cells. We also measured pre-mRNA accumulation of the meiotic MER2 gene, which depends on the expression of Mer1 factor for splicing. prp45(1-169) cells accumulated approximately threefold higher levels of MER2 pre-mRNA than WT cells only when splicing was induced. To monitor cotranscriptional splicing, we determined the presence of early spliceosome assembly factors and snRNP complexes along the ECM33 and ACT1 genes. We found that prp45(1-169) hampered the cotranscriptional recruitment of U2 and, to a larger extent, U5 and NTC, while the U1 profile was unaffected. The recruitment of Prp45(1-169) was impaired similarly to U5 snRNP and NTC. Our results imply that Prp45 is required for timely formation of complex A, prior to stable physical association of U5/NTC with the emerging pre-mRNA substrate. We suggest that Prp45 facilitates conformational rearrangements and/or contacts that couple U1 snRNP-recognition to downstream assembly events.

• Klíčová slova
• Prp8, RES complex, chromatin immunoprecipitation, cotranscriptional splicing, nineteen complex, spliceosome assembly,
• MeSH
• introny MeSH
• malý jaderný ribonukleoprotein U1 metabolismus   MeSH
• malý jaderný ribonukleoprotein U2 metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sestřih RNA * MeSH
• spliceozomy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U1 MeSH
• malý jaderný ribonukleoprotein U2 MeSH
• PRP45 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

Although atomistic explicit-solvent Molecular Dynamics (MD) is a popular tool to study protein-RNA recognition, satisfactory MD description of protein-RNA complexes is not always achieved. Unfortunately, it is often difficult to separate MD simulation instabilities primarily caused by the simple point-charge molecular mechanics (MM) force fields from problems related to the notorious uncertainties in the starting structures. Herein, we report a series of large-scale QM/MM calculations on the U1A protein-RNA complex. This experimentally well-characterized system has an intricate protein-RNA interface, which is very unstable in MD simulations. The QM/MM calculations identify several H-bonds poorly described by the MM method and thus indicate the sources of instabilities of the U1A interface in MD simulations. The results suggest that advanced QM/MM computations could be used to indirectly rationalize problems seen in MM-based MD simulations of protein-RNA complexes. As the most accurate QM method, we employ the computationally demanding meta-GGA density functional TPSS-D3(BJ)/def2-TZVP level of theory. Because considerably faster methods would be needed to extend sampling and to study even larger protein-RNA interfaces, a set of low-cost QM/MM methods is compared to the TPSS-D3(BJ)/def2-TZVP data. The PBEh-3c and B97-3c density functional composite methods appear to be suitable for protein-RNA interfaces. In contrast, HF-3c and the tight-binding Hamiltonians DFTB3-D3 and GFN-xTB perform unsatisfactorily and do not provide any advantage over the MM description. These conclusions are supported also by similar analysis of a simple HutP protein-RNA interface, which is well-described by MD with the exception of just one H-bond. Some other methodological aspects of QM/MM calculations on protein-RNA interfaces are discussed.

• MeSH
• Bacillus subtilis chemie   metabolismus   MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• kvantová teorie MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U1 chemie   metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA chemie   metabolismus   MeSH
• simulace molekulární dynamiky * ekonomika   MeSH
• software MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• malý jaderný ribonukleoprotein U1 MeSH
• proteiny vázající RNA MeSH
• RNA MeSH
• U1A protein MeSH Prohlížeč

Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.

• MeSH
• exony * MeSH
• HeLa buňky MeSH
• lidé MeSH
• místa sestřihu RNA * MeSH
• RNA malá jaderná * metabolismus   genetika   MeSH
• sekvence nukleotidů MeSH
• sestřih RNA MeSH
• transkripční faktor STAT3 * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• místa sestřihu RNA * MeSH
• RNA malá jaderná * MeSH
• STAT3 protein, human MeSH Prohlížeč
• transkripční faktor STAT3 * MeSH
• U1 small nuclear RNA MeSH Prohlížeč
• U6 small nuclear RNA MeSH Prohlížeč

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3' splice sites (3'ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3'ss and branch points of a PUF60-dependent exon and the 3'ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3'ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

• MeSH
• aminokyselinové motivy MeSH
• exony * MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• heterogenní jaderné ribonukleoproteiny metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• krátké rozptýlené jaderné elementy MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U1 metabolismus   MeSH
• missense mutace * MeSH
• místa sestřihu RNA * MeSH
• proteiny vázající RNA metabolismus   MeSH
• represorové proteiny chemie   nedostatek   metabolismus   MeSH
• sekvenční analýza RNA MeSH
• sestřihové faktory chemie   nedostatek   metabolismus   MeSH
• sestřihový faktor U2AF MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HCC1 autoantigen MeSH Prohlížeč
• heterogenní jaderné ribonukleoproteiny MeSH
• jaderné proteiny MeSH
• malý jaderný ribonukleoprotein U1 MeSH
• místa sestřihu RNA * MeSH
• poly-U binding splicing factor 60KDa MeSH Prohlížeč
• proteiny vázající RNA MeSH
• represorové proteiny MeSH
• sestřihové faktory MeSH
• sestřihový faktor U2AF MeSH
• SNRNP70 protein, human MeSH Prohlížeč

Splicing is catalyzed by the spliceosome, a complex of five major small nuclear ribonucleoprotein particles (snRNPs). The pre-mRNA splicing factor PRPF8 is a crucial component of the U5 snRNP, and together with EFTUD2 and SNRNP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFTUD2, and SNRNP200 in association with the HSP90/R2TP complex, its ZNHIT2 cofactor, and additional proteins. HSP90 and R2TP bind unassembled U5 proteins in the cytoplasm, stabilize them, and promote the formation of the U5 snRNP. We further found that PRPF8 mutants causing Retinitis pigmentosa assemble less efficiently with the U5 snRNP and bind more strongly to R2TP, with one mutant retained in the cytoplasm in an R2TP-dependent manner. We propose that the HSP90/R2TP chaperone system promotes the assembly of a key module of U5 snRNP while assuring the quality control of PRPF8. The proteomics data further reveal new interactions between R2TP and the tuberous sclerosis complex (TSC), pointing to a potential link between growth signals and the assembly of key cellular machines.

• MeSH
• elongační faktory genetika   metabolismus   MeSH
• HeLa buňky MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U1 metabolismus   MeSH
• malý jaderný ribonukleoprotein U4-U6 metabolismus   MeSH
• malý jaderný ribonukleoprotein U5 genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• multiproteinové komplexy MeSH
• mutace MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• proteiny vázající vápník metabolismus   MeSH
• proteomika metody   MeSH
• retinopathia pigmentosa genetika   metabolismus   MeSH
• RNA interference MeSH
• sestřih RNA * MeSH
• stabilita proteinů MeSH
• transfekce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• EFTUD2 protein, human MeSH Prohlížeč
• elongační faktory MeSH
• malý jaderný ribonukleoprotein U1 MeSH
• malý jaderný ribonukleoprotein U4-U6 MeSH
• malý jaderný ribonukleoprotein U5 MeSH
• messenger RNA MeSH
• multiproteinové komplexy MeSH
• prekurzory RNA MeSH
• proteiny tepelného šoku HSP90 MeSH
• proteiny vázající RNA MeSH
• proteiny vázající vápník MeSH
• PRPF8 protein, human MeSH Prohlížeč
• TESC protein, human MeSH Prohlížeč

By virtue of its preferential binding to poly(U) tails on small RNA precursors and nuclear localisation motif, the La protein has been implicated for a role in the stabilisation and nuclear retention of processing intermediates for a variety of small RNAs in eukaryotic cells. As the universal substrate for trans-splicing, the spliced leader RNA is transcribed as a precursor with just such a tail. La protein was targeted for selective knockdown by inducible RNA interference in Trypanosoma brucei. Of three RNA interference strategies employed, a p2T7-177 vector was the most effective in reducing both the La mRNA as well as the protein itself from induced cells. In the relative absence of La protein T. brucei cells were not viable, in contrast to La gene knockouts in yeast. A variety of potential small RNA substrates were examined under induction, including spliced leader RNA, spliced leader associated RNA, the U1, U2, U4, and U6 small nuclear RNAs, 5S ribosomal RNA, U3 small nucleolar RNA, and tRNATyr. None of these molecules showed significant variance in size or abundance in their mature forms, although a discrete subset of intermediates appear for spliced leader RNA and tRNATyr intron splicing under La depletion conditions. 5'-end methylation in the spliced leader RNA and U1 small nuclear RNA was unaffected. The immediate cause of lethality in T. brucei was not apparent, but may represent a cumulative effect of multiple defects including processing of spliced leader RNA, tRNATyr and other unidentified RNA substrates. This study indicates that La protein binding is not essential for maturation of the spliced leader RNA, but does not rule out the presence of an alternative processing pathway that could compensate for the absence of normally-associated La protein.

• MeSH
• malá interferující RNA genetika   MeSH
• protozoální geny * MeSH
• protozoální proteiny genetika   MeSH
• RNA malá jaderná genetika   MeSH
• RNA se sestřihovou vedoucí sekvencí genetika   MeSH
• RNA transferová genetika   MeSH
• sestřih RNA MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• malá interferující RNA MeSH
• protozoální proteiny MeSH
• RNA malá jaderná MeSH
• RNA se sestřihovou vedoucí sekvencí MeSH
• RNA transferová MeSH

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre- and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2'-oxygens near the scissile phosphate compromise catalytic in-line fitness. A cis-acting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape.

• Klíčová slova
• conformational change, molecular dynamics simulation, small ribozyme, steady-state FRET, time-resolved FRET,
• MeSH
• katalytická doména MeSH
• katalýza MeSH
• kinetika MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• RNA katalytická chemie   MeSH
• RNA malá jaderná chemie   metabolismus   MeSH
• RNA virová chemie   MeSH
• simulace molekulární dynamiky MeSH
• štěpení RNA MeSH
• virus hepatitidy delta enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• hammerhead ribozyme MeSH Prohlížeč
• RNA katalytická MeSH
• RNA malá jaderná MeSH
• RNA virová MeSH
• U1 small nuclear RNA MeSH Prohlížeč

Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.

• Klíčová slova
• 5′ss, SMN1, U1 snRNA, cryptic splice sites, splicing-affecting mutation,
• MeSH
• alternativní sestřih * MeSH
• buněčné linie MeSH
• exony * MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• místa sestřihu RNA MeSH
• mutace * MeSH
• mutageneze MeSH
• protein přežití motorických neuronů 1 chemie   genetika   metabolismus   MeSH
• RNA malá jaderná chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• místa sestřihu RNA MeSH
• protein přežití motorických neuronů 1 MeSH
• RNA malá jaderná MeSH
• SMN1 protein, human MeSH Prohlížeč
• U1 small nuclear RNA MeSH Prohlížeč

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

• MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• fluorescenční spektrometrie MeSH
• FRAP MeSH
• HeLa buňky MeSH
• kinetika MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• prekurzory RNA metabolismus   MeSH
• ribonukleoproteiny malé jaderné metabolismus   fyziologie   MeSH
• sestřih RNA fyziologie   MeSH
• spliceozomy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• prekurzory RNA MeSH
• ribonukleoproteiny malé jaderné MeSH