The nucleus of higher eukaryotes contains a number of structures that concentrate specific biomolecules and play distinct roles in nuclear metabolism. In recent years, the molecular mechanisms controlling their formation have been intensively studied. In this brief review, I focus on coilin and Cajal bodies. Coilin is a key scaffolding protein of Cajal bodies that is evolutionarily conserved in metazoans. Cajal bodies are thought to be one of the archetypal nuclear structures involved in the metabolism of several short non-coding nuclear RNAs. Yet surprisingly little is known about the structure and function of coilin, and a comprehensive model to explain the origin of Cajal bodies is also lacking. Here, I summarize recent results on Cajal bodies and coilin and discuss them in the context of the last three decades of research in this field.

• Klíčová slova
• Cajal bodies, coilin, snRNA, snRNP, snoRNA, telomerase RNA,
• MeSH
• buněčné jádro * MeSH
• Cajalova tělíska * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Spliceosomal snRNPs are multicomponent particles that undergo a complex maturation pathway. Human Sm-class snRNAs are generated as 3'-end extended precursors, which are exported to the cytoplasm and assembled together with Sm proteins into core RNPs by the SMN complex. Here, we provide evidence that these pre-snRNA substrates contain compact, evolutionarily conserved secondary structures that overlap with the Sm binding site. These structural motifs in pre-snRNAs are predicted to interfere with Sm core assembly. We model structural rearrangements that lead to an open pre-snRNA conformation compatible with Sm protein interaction. The predicted rearrangement pathway is conserved in Metazoa and requires an external factor that initiates snRNA remodeling. We show that the essential helicase Gemin3, which is a component of the SMN complex, is crucial for snRNA structural rearrangements during snRNP maturation. The SMN complex thus facilitates ATP-driven structural changes in snRNAs that expose the Sm site and enable Sm protein binding.

• MeSH
• HeLa buňky MeSH
• jádro snRNP - proteiny genetika   MeSH
• lidé MeSH
• prekurzory RNA * metabolismus   MeSH
• proteinový komplex SMN metabolismus   MeSH
• ribonukleoproteiny malé jaderné metabolismus   MeSH
• RNA malá jaderná * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• jádro snRNP - proteiny MeSH
• prekurzory RNA * MeSH
• proteinový komplex SMN MeSH
• ribonukleoproteiny malé jaderné MeSH
• RNA malá jaderná * MeSH

Coilin is a conserved protein essential for integrity of nuclear membrane-less inclusions called Cajal bodies. Here, we report an amino acid substitution (p.K496E) found in a widely-used human EGFP-coilin construct that has a dominant-negative effect on Cajal body formation. We show that this coilin-K496E variant fails to rescue Cajal bodies in cells lacking endogenous coilin, whereas the wild-type construct restores Cajal bodies in mouse and human coilin-knockout cells. In cells containing endogenous coilin, both the wild-type and K496E variant proteins accumulate in Cajal bodies. However, high-level overexpression of coilin-K496E causes Cajal body disintegration. Thus, a mutation in the C-terminal region of human coilin can disrupt Cajal body assembly. Caution should be used when interpreting data from coilin plasmids that are derived from this variant (currently deposited at Addgene).

• Klíčová slova
• Cajal bodies, Coilin, Mutation,
• MeSH
• bodová mutace * genetika   MeSH
• Cajalova tělíska * genetika   MeSH
• HeLa buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mutace genetika   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• jaderné proteiny MeSH

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo a complex maturation pathway containing multiple steps in the nucleus and in the cytoplasm. snRNP biogenesis is strictly proofread and several quality control checkpoints are placed along the pathway. Here, we analyzed the fate of small nuclear RNAs (snRNAs) that are unable to acquire a ring of Sm proteins. We showed that snRNAs lacking the Sm ring are unstable and accumulate in P-bodies in an LSm1-dependent manner. We further provide evidence that defective snRNAs without the Sm binding site are uridylated at the 3' end and associate with DIS3L2 3'→5' exoribonuclease and LSm proteins. Finally, inhibition of 5'→3' exoribonuclease XRN1 increases association of ΔSm snRNAs with DIS3L2, which indicates competition and compensation between these two degradation enzymes. Together, we provide evidence that defective snRNAs without the Sm ring are uridylated and degraded by alternative pathways involving either DIS3L2 or LSm proteins and XRN1.

• MeSH
• exoribonukleasy metabolismus   MeSH
• HeLa buňky MeSH
• konformace nukleové kyseliny * MeSH
• lidé MeSH
• organely metabolismus   MeSH
• proteinový komplex SMN metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• RNA malá jaderná chemie   metabolismus   MeSH
• sekvence nukleotidů MeSH
• stabilita RNA MeSH
• transport RNA * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DIS3L2 protein, human MeSH Prohlížeč
• exoribonukleasy MeSH
• GEMIN5 protein, human MeSH Prohlížeč
• LSM1 protein, human MeSH Prohlížeč
• proteinový komplex SMN MeSH
• proteiny vázající RNA MeSH
• protoonkogenní proteiny MeSH
• RNA malá jaderná MeSH

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

Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.

• MeSH
• buněčné jádro genetika   MeSH
• Cajalova tělíska genetika   metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U2 genetika   MeSH
• regulace genové exprese genetika   MeSH
• RNA malá jaderná genetika   MeSH
• spliceozomy genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U2 MeSH
• RNA malá jaderná MeSH
• U2 small nuclear RNA MeSH Prohlížeč

Spliceosomal snRNPs are complex particles that proceed through a fascinating maturation pathway. Several steps of this pathway are closely linked to nuclear non-membrane structures called Cajal bodies. In this review, I summarize the last 20 y of research in this field. I primarily focus on snRNP biogenesis, specifically on the steps that involve Cajal bodies. I also evaluate the contribution of the Cajal body in snRNP quality control and discuss the role of snRNPs in Cajal body formation.

• MeSH
• Cajalova tělíska metabolismus   MeSH
• genetická transkripce MeSH
• lidé MeSH
• posttranskripční úpravy RNA MeSH
• ribonukleoproteiny malé jaderné genetika   metabolismus   MeSH
• spliceozomy MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ribonukleoproteiny malé jaderné MeSH

The mechanical stability of epithelial cells, which protect organisms from harmful external factors, is maintained by hemidesmosomes via the interaction between plectin 1a (P1a) and integrin α6β4. Binding of calcium-calmodulin (Ca(2+)-CaM) to P1a together with phosphorylation of integrin β4 disrupts this complex, resulting in disassembly of hemidesmosomes. We present structures of the P1a actin binding domain either in complex with the N-ter lobe of Ca(2+)-CaM or with the first pair of integrin β4 fibronectin domains. Ca(2+)-CaM binds to the N-ter isoform-specific tail of P1a in a unique manner, via its N-ter lobe in an extended conformation. Structural, cell biology, and biochemical studies suggest the following model: binding of Ca(2+)-CaM to an intrinsically disordered N-ter segment of plectin converts it to an α helix, which repositions calmodulin to displace integrin β4 by steric repulsion. This model could serve as a blueprint for studies aimed at understanding how Ca(2+)-CaM or EF-hand motifs regulate F-actin-based cytoskeleton.

• MeSH
• hemidesmozomy chemie   MeSH
• hydrofobní a hydrofilní interakce MeSH
• integrin beta4 chemie   MeSH
• interakční proteinové domény a motivy MeSH
• kalmodulin chemie   MeSH
• krysa rodu Rattus MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• plektin chemie   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• integrin beta4 MeSH
• kalmodulin MeSH
• Plec protein, mouse MeSH Prohlížeč
• plektin MeSH

Förster resonance energy transfer (FRET) microscopy is a powerful technique routinely used to monitor interactions between biomolecules. Here, we focus on the techniques that are used for investigating the structure and interactions of nucleic acids (NAs). We present a brief overview of the most commonly used FRET microscopy techniques, their advantages and drawbacks. We list experimental approaches recently used for either in vitro or in vivo studies. Next, we summarize how FRET contributed to the understanding of pre-mRNA splicing and spliceosome assembly.

• MeSH
• alternativní sestřih MeSH
• fluorescenční mikroskopie * MeSH
• molekulární konformace MeSH
• nukleoproteiny metabolismus   MeSH
• nukleové kyseliny chemie   genetika   metabolismus   MeSH
• prekurzory RNA chemie   genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie * MeSH
• sestřih RNA * MeSH
• spliceozomy genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• nukleoproteiny MeSH
• nukleové kyseliny MeSH
• prekurzory RNA MeSH

Processing bodies (P-bodies) are dynamic cytoplasmic structures involved in mRNA degradation, but the mechanism that governs their formation is poorly understood. In this paper, we address a role of Like-Sm (LSm) proteins in formation of P-bodies and provide evidence that depletion of nuclear LSm8 increases the number of P-bodies, while LSm8 overexpression leads to P-body loss. We show that LSm8 knockdown causes relocalization of LSm4 and LSm6 proteins to the cytoplasm and suggest that LSm8 controls nuclear accumulation of all LSm2-7 proteins. We propose a model in which redistribution of LSm2-7 to the cytoplasm creates new binding sites for other P-body components and nucleates new, microscopically visible structures. The model is supported by prolonged residence of two P-body proteins, DDX6 and Ago2, in P-bodies after LSm8 depletion, which indicates stronger interactions between these proteins and P-bodies. Finally, an increased number of P-bodies has negligible effects on microRNA-mediated translation repression and nonsense mediated decay, further supporting the view that the function of proteins localized in P-bodies is independent of visible P-bodies.

• MeSH
• autoantigeny metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• cytoplazmatická granula metabolismus   MeSH
• DEAD-box RNA-helikasy metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U4-U6 metabolismus   fyziologie   MeSH
• N-terminální acetyltransferasa C metabolismus   fyziologie   MeSH
• posttranskripční úpravy RNA * MeSH
• proteiny vázající RNA metabolismus   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• ribonukleoproteiny malé jaderné metabolismus   MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoantigeny MeSH
• DDX6 protein, human MeSH Prohlížeč
• DEAD-box RNA-helikasy MeSH
• malý jaderný ribonukleoprotein U4-U6 MeSH
• N-terminální acetyltransferasa C MeSH
• NAA38 protein, human MeSH Prohlížeč
• proteiny vázající RNA MeSH
• protoonkogenní proteiny MeSH
• rekombinantní fúzní proteiny MeSH
• ribonukleoproteiny malé jaderné MeSH
• TNRC6A protein, human MeSH Prohlížeč