scholarly journals Conserved Stem II of the Box C/D Motif Is Essential for Nucleolar Localization and Is Required, Along with the 15.5K Protein, for the Hierarchical Assembly of the Box C/D snoRNP

2002 ◽  
Vol 22 (23) ◽  
pp. 8342-8352 ◽  
Author(s):  
Nicholas J. Watkins ◽  
Achim Dickmanns ◽  
Reinhard Lührmann
Keyword(s):  
Specific Binding ◽  
Nucleolar Localization ◽  
Homologous Proteins ◽  
Stem Loop ◽  
Hierarchical Assembly ◽  
Conserved Sequence ◽  
Associated Proteins ◽  
The Individual ◽  
Stem Structure

ABSTRACT The 5′ stem-loop of the U4 snRNA and the box C/D motif of the box C/D snoRNAs can both be folded into a similar stem-internal loop-stem structure that binds the 15.5K protein. The homologous proteins NOP56 and NOP58 and 61K (hPrp31) associate with the box C/D snoRNPs and the U4/U6 snRNP, respectively. This raises the intriguing question of how the two homologous RNP complexes specifically assemble onto similar RNAs. Here we investigate the requirements for the specific binding of the individual snoRNP proteins to the U14 box C/D snoRNPs in vitro. This revealed that the binding of 15.5K to the box C/D motif is essential for the association of the remaining snoRNP-associated proteins, namely, NOP56, NOP58, fibrillarin, and the nucleoplasmic proteins TIP48 and TIP49. Stem II of the box C/D motif, in contrast to the U4 5′ stem-loop, is highly conserved, and we show that this sequence is responsible for the binding of NOP56, NOP58, fibrillarin, TIP48, and TIP49, but not of 15.5K, to the snoRNA. Indeed, the sequence of stem II was essential for nucleolar localization of U14 snoRNA microinjected into HeLa cells. Thus, the conserved sequence of stem II determines the specific assembly of the box C/D snoRNP.

scholarly journals Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1517
Author(s):  
Rebecca S. Brown ◽  
Lisa Kim ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Rna Structure ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Specific Binding ◽  
Genomic Rna ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Labeled Rna

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

scholarly journals Different Mechanisms for Pseudouridine Formation in Yeast 5S and 5.8S rRNAs

2008 ◽  
Vol 28 (10) ◽  
pp. 3089-3100 ◽  
Author(s):  
Wayne A. Decatur ◽  
Murray N. Schnare
Keyword(s):  
Large Subunit ◽  
Consensus Sequence ◽  
5S Rrna ◽  
Stem Loop ◽  
Conserved Sequence ◽  
5.8S Rrna ◽  
Sequence Elements ◽  
The Individual ◽  
A Site ◽  
Large Subunit Rrna

ABSTRACT The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Ψs) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Ψ formation in 5S and 5.8S rRNA in which the unassigned Ψs occur. We show that while the formation of the Ψ in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Ψ in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Ψ in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.

scholarly journals Revealing the high propensity of RNAs to non-specifically bind drug-like small molecules

2020 ◽  
Author(s):  
Megan L. Kelly ◽  
Chia-Chieh Chu ◽  
Honglue Shi ◽  
Laura R. Ganser ◽  
Hal P. Bogerd ◽  
...  
Keyword(s):  
Small Molecules ◽  
Specific Binding ◽  
Cellular Activity ◽  
Response Element ◽  
Stem Loop ◽  
Rev Response Element ◽  
Nmr Structures ◽  
Important Challenge ◽  
Rna Binders

ABSTRACTIdentifying small molecules that selectively bind a single RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to non-specific binding of aminoglycosides and intercalators to a variety of RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities, however the ability of such compounds to discriminate against RNA stem-loops commonly found in the transcriptome has not been thoroughly assessed in all cases. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activity of three distinct RNAs, to non-specifically bind two HIV-1 stem-loop RNAs: the transactivation response element (TAR) and stem IIB in the rev response element (RREIIB). All three compounds bound to TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting substantial off-target interactions consistent with non-specific cellular activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, much like aminoglycosides, and in contrast to ligands that specifically bind riboswitches. Our results support extending the group of non-selective RNA-binders beyond aminoglycosides and intercalators to encompass drug-like compounds with capacity for non-specific hydrogen-bonding and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

scholarly journals Host poly(A) polymerases PAPD5 and PAPD7 provide two layers of protection that ensure the integrity and stability of hepatitis B virus RNA

2021 ◽  
Author(s):  
Fei Liu ◽  
Amy C.H Lee ◽  
Fang Guo ◽  
Andrew S. Kondratowicz ◽  
Holly M Micolochick Steuer ◽  
...  
Keyword(s):  
Dominant Role ◽  
Regulatory Element ◽  
Rna Stability ◽  
Antiviral Effect ◽  
Inhibitory Effect ◽  
Stem Loop ◽  
Knock Out ◽  
The Individual

Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize HBV RNA via the interaction with the viral post-transcriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, HBsAg production and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies, therefore it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (EC50 ranged from 1.4 to 6.8 nM) and in vivo by 0.93 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity towards the inhibitory effect of AB-452. Although neither single knock-out (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO could not result in any measurable phenotypic changes, but displays a similar antiviral effect as AB-452 treatment when PAPD5 is depleted simultaneously. PAPD5/7 double KO confers complete resistance to AB-452 treatment. Our results thus indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5 targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication.

scholarly journals Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways

2001 ◽  
Vol 21 (23) ◽  
pp. 8035-8044 ◽  
Author(s):  
Daphné Seigneurin-Berny ◽  
André Verdel ◽  
Sandrine Curtet ◽  
Claudie Lemercier ◽  
Jérôme Garin ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Specific Binding ◽  
Protein A ◽  
Biochemical Properties ◽  
Mouse Testis ◽  
Histone Deacetylase 6 ◽  
Protein Ubiquitination ◽  
Ubiquitin Binding ◽  
Associated Proteins

ABSTRACT The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

scholarly journals The specific binding of the microtubule-associated protein 2 (MAP2) to the outer membrane of rat brain mitochondria

1989 ◽  
Vol 261 (1) ◽  
pp. 167-173 ◽  
Author(s):  
M Lindén ◽  
B D Nelson ◽  
J F Leterrier
Keyword(s):  
Rat Brain ◽  
Outer Membrane ◽  
Binding Sites ◽  
Specific Binding ◽  
Brain Mitochondria ◽  
Microtubule Associated Proteins ◽  
Cross Linkage ◽  
Associated Proteins ◽  
A Site

Purified mitochondria from rat brain contain microtubule-associated proteins (MAPs) bound to the outer membrane. Studies of binding in vitro performed with microtubules and with purified microtubule proteins showed that mitochondria preferentially interact with the high-molecular-mass MAPs (and not with Tau protein). Incubation of intact mitochondria with Taxol-stabilized microtubules resulted in the selective trapping of both MAPs 1 and 2 on mitochondria, indicating that an interaction between the two organelles occurred through a site on the arm-like projection of MAPs. Two MAP-binding sites were located on intact mitochondria. The lower-affinity MAP2-binding site (Kd = 2 x 10(-7) M) was preserved and enriched in the outer-membrane fraction, whereas the higher-affinity site (Kd = 1 x 10(-9) M) was destroyed after removing the outer membrane with digitonin. Detergent fractionation of mitochondrial outer membranes saturated with MAP2 bound in vitro showed that MAPs are associated with membrane fragments which contain the pore-forming protein (porin). MAP2 also partially prevents the solubilization of porin from outer membrane, indicating a MAP-induced change in the membrane environment of porin. These observations demonstrate the presence of specific MAP-binding sites on the outer membrane, suggesting an association between porin and the membrane domain involved in the cross-linkage between microtubules and mitochondria.

scholarly journals A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

1989 ◽  
Vol 9 (11) ◽  
pp. 4872-4881 ◽  
Author(s):  
C C Query ◽  
R C Bentley ◽  
J D Keene
Keyword(s):  
Nucleotide Sequence ◽  
Splice Site ◽  
Rna Binding ◽  
Binding Domain ◽  
Specific Domain ◽  
Stem Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Associated Proteins ◽  
Nuclear Ribonucleoprotein

We have defined the nucleotide sequence of a protein-binding domain within U1 RNA that specifically recognizes and binds both to a U1 small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified U1 RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of U1 RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of U1 RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of U1 RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.

scholarly journals The U1 RNA-binding site of the U1 small nuclear ribonucleoprotein (snRNP)-associated A protein suggests a similarity with U2 snRNPs.

1989 ◽  
Vol 9 (7) ◽  
pp. 2975-2982 ◽  
Author(s):  
C Lutz-Freyermuth ◽  
J D Keene ◽  
C Lutz-Reyermuth
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Sequence Similarity ◽  
Binding Assays ◽  
Stem Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Vitro Binding ◽  
Associated Proteins ◽  
Nuclear Ribonucleoprotein

The site of interaction between human U1 RNA and one of its uniquely associated proteins, A, was examined with in vitro binding assays. The A protein bound directly to stem-loop II of U1 RNA in a region which exhibits sequence similarity to U2 RNA. The similarity with U2 RNA was in a region that has been shown to interact with a U2 RNA-associated protein. The A protein-binding site on U1 RNA overlapped a previously described epitope for an RNA-specific human autoantibody (S. L. Deutscher and J. D. Keene, Proc. Natl. Acad. Sci. USA 85:3299-3303, 1988), supporting the hypothesis that the anti-RNA antibody originated as an anti-idiotypic response to A protein-specific autoantibodies.

scholarly journals Divergence in Thermostability of Arabidopsis Mitochondrial Nucleotide Exchange Factors Encoded by Duplicate Genes, MGE1 and MGE2

2019 ◽  
Author(s):  
Zih-teng Chen ◽  
Meng-Ju Hung ◽  
Shih-Jiun Yu ◽  
Tai-Yan Liao ◽  
Yao-Pin Lin ◽  
...  
Keyword(s):  
Elevated Temperatures ◽  
Duplication Event ◽  
Duplicate Genes ◽  
Nucleotide Exchange ◽  
Homologous Proteins ◽  
Genome Duplication Event ◽  
E Coli ◽  
Conserved Sequence ◽  
Nucleotide Exchange Factors

ABSTRACTThe divergence of duplicate genes links to organismic adaptation. In Arabidopsis thaliana two nuclear genes encode mitochondrial GrpEs, MGE1 and MGE2, the nucleotide exchange factors of DnaK/HSP70 chaperone. MGE1 and MGE2 are duplicate genes originated from a whole genome duplication event. They respond differentially to high temperature; MGE2 is heat-inducible and is required for Arabidopsis seedlings to tolerate prolonged heat stress, while MGE1 is constitutively expressed. Heterologous expression of MGE2 but not MGE1 restored the growth of E. coli grpE mutant cells at elevated temperatures, suggesting that MGE2 is more thermostable than MGE1. In this study, we directly compared the thermostability of the purified recombinant MGE1 and MGE2 by circular dichroism spectroscopy. The temperature midpoints of the unfolding transition (Tm) of MGE1 and MGE2 were about 38 and 46 °C, respectively, indicating that MGE2 is remarkably more stable than MGE1 at higher temperature. Domain swapping between the two homologous proteins showed that the N-terminal region, including an unstructured sequence and a long α-helix domain, is the major determinant of the thermostability. Although MGE2 contains a conserved sequence derived from an exonized intron within the N-terminus unstructured region, deletion of this sequence did not substantially affect protein thermostability in vitro and complementation of E. coli and Arabidopsis heat sensitive mutants. Taken together, our results suggest that Arabidopsis MGE1 and MGE2 had diverged not only in transcriptional response but also in the thermostability of the encoded proteins, which may contribute to adaptation of plants to higher temperatures.

Purification of developmentally regulated avian 400-kDa intermediate filament associated protein. Molecular interactions with intermediate filament proteins and other cytoskeleton components

1995 ◽  
Vol 73 (9-10) ◽  
pp. 651-657 ◽  
Author(s):  
Marie Duval ◽  
Xiaoying Ma ◽  
Jean-Paul Valet ◽  
Michel Vincent
Keyword(s):  
Chick Embryo ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Specific Binding ◽  
Developmentally Regulated ◽  
Intermediate Filament Proteins ◽  
In Vitro Recombination ◽  
Associated Proteins ◽  
Structural Lattice

IFAPa-400, a 400-kDa developmentally regulated protein thought to be associated with intermediate filaments, has been purified from chick embryo hearts to investigate its interaction with vimentin and other IF proteins and to identify other cellular components to which this cytoskeletal protein associates. Previous studies suggested that this protein was associated with the vimentin-containing intermediate filament lattice of myoblasts and neuroblasts before their terminal differentiation, providing these cells with a particular intermediate filament cytoskeleton that could satisfy specific mechanical requirements during their intense morphogenetic activities. Although IFAPa-400 partially reassociated with vimentin and desmin in disassembly–reassembly experiments using crude IF preparations from chick embryo hearts, in vitro recombination of purified IFAPa-400 with vimentin and desmin failed to demonstrate any direct association. When purified IFAPa-400 was used as a probe in blot overlay assays, however, specific binding to vimentin and desmin was observed, providing the first evidence of a physical association between IFAPa-400 and intermediate filament proteins. The blot overlay experiments also demonstrated that IFAPa-400 binds to two unidentified polypeptides of 19 and 32 kDa. These results are thus consistent with the hypothesis that a structural lattice requiring a vimentin–IFAPa-400 combination constitutes the intermediate filament system of myogenic and neurogenic cells.Key words: cytoskeleton, intermediate filaments, intermediate filament associated proteins, vimentin, IFAPa-400.

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