RNP Structure Research Articles

The RES complex is required for efficient transformation of the precatalytic B spliceosome into an activated Bact complex.

The precise function of the trimeric retention and splicing (RES) complex in pre-mRNA splicing remains unclear. Here we dissected the role of RES during the assembly and activation of yeast spliceosomes. The efficiency of pre-mRNA splicing was significantly lower in the absence of the RES protein Snu17, and the recruitment of its binding partners, Pml1 (pre-mRNA leakage protein 1) and Bud13 (bud site selection protein 13), to the spliceosome was either abolished or substantially reduced. RES was not required for the assembly of spliceosomal B complexes, but its absence hindered efficient Bact complex formation. ΔRES spliceosomes were no longer strictly dependent on Prp2 activity for their catalytic activation, suggesting that they are structurally compromised. Addition of Prp2, Spp2, and UTP to affinity-purified ΔRES B or a mixture of B/Bact complexes formed on wild-type pre-mRNA led to their disassembly. However, no substantial disassembly was observed with ΔRES spliceosomes formed on a truncated pre-mRNA that allows Prp2 binding but blocks its activity. Thus, in the absence of RES, Prp2 appears to bind prematurely, leading to the disassembly of the ΔRES B complexes to which it binds. Our data suggest that Prp2 can dismantle B complexes with an aberrant protein composition, suggesting that it may proofread the spliceosome's RNP structure prior to activation.

Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2.

The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1΄s G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43΄s cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1΄s C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as ‘doorkeepers’ and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly.

Composition of yeast snRNPs and snoRNPs in the absence of trimethylguanosine caps reveals nuclear cap binding protein as a gained U1 component implicated in the cold-sensitivity of tgs1Δ cells

Small nuclear and nucleolar RNAs that program pre-mRNA splicing and rRNA processing have a signature 5′-trimethylguanosine (TMG) cap. Whereas the mechanism of TMG synthesis by Tgs1 methyltransferase has been elucidated, we know little about whether or how RNP biogenesis, structure and function are perturbed when TMG caps are missing. Here, we analyzed RNPs isolated by tandem-affinity purification from TGS1 and tgs1Δ yeast strains. The protein and U-RNA contents of total SmB-containing RNPs were similar. Finer analysis revealed stoichiometric association of the nuclear cap-binding protein (CBP) subunits Sto1 and Cbc2 with otherwise intact Mud1- and Nam8-containing U1 snRNPs from tgs1Δ cells. CBP was not comparably enriched in Lea1-containing U2 snRNPs from tgs1Δ cells. Moreover, CBP was not associated with mature Nop58-containing C/D snoRNPs or mature Cbf5- and Gar1-containing H/ACA snoRNPs from tgs1Δ cells. The protein composition and association of C/D snoRNPs with the small subunit (SSU) processosome were not grossly affected by absence of TMG caps, nor was the composition of H/ACA snoRNPs. The cold-sensitive (cs) growth defect of tgs1Δ yeast cells could be suppressed by mutating the cap-binding pocket of Cbc2, suggesting that ectopic CBP binding to the exposed U1 m7G cap in tgs1Δ cells (not lack of TMG caps per se) underlies the cs phenotype.

Structural Basis for Substrate Placement by an Archaeal Box C/D Ribonucleoprotein Particle

Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2'-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a half-mer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modeling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs.

Structure function studies on different structural domains of nucleoprotein of H1N1 subtype

Recent 2009 flu pandemic is a global outbreak of a new strain of influenza A virus subtype H1N1. The H1N1 virus has crossed species barrier to human and apparently acquired the capability to transmit this disease from human to human. The NP is a multifunctional protein that not only encapsidates viral RNA (vRNA), but also forms homo-oligomer and thereby maintains RNP structure. It is also thought to be the key adaptor for virus and host cell interaction. Thus, it is one of the factor that play a key role in the pathogenesis of influenza A virus infection. Therefore, to understand the cause of pathogenicity of H1N1 virus, we have studied the structure-function relationship of different domains of NP. Our results showed that conservative mutation in NP of various strains were pathogenic in nature. However, non-conservative mutation slightly abrogated oligomerization and was therefore less pathogenic. Our results also suggest that beside tail and body domain, head domain may also participate in an oligomerization process.

The coiled-coil domain of the Nop56/58 core protein is dispensable for sRNP assembly but is critical for archaeal box C/D sRNP-guided nucleotide methylation

Archaeal box C/D sRNAs guide the methylation of specific nucleotides in archaeal ribosomal and tRNAs. Three Methanocaldococcus jannaschii sRNP core proteins (ribosomal protein L7, Nop56/58, and fibrillarin) bind the box C/D sRNAs to assemble the sRNP complex, and these core proteins are essential for nucleotide methylation. A distinguishing feature of the Nop56/58 core protein is the coiled-coil domain, established by alpha-helices 4 and 5, that facilitates Nop56/58 self-dimerization in vitro. The function of this coiled-coil domain has been assessed for box C/D sRNP assembly, sRNP structure, and sRNP-guided nucleotide methylation by mutating or deleting this protein domain. Protein pull-down experiments demonstrated that Nop56/58 self-dimerization and Nop56/58 dimerization with the core protein fibrillarin are mutually exclusive protein:protein interactions. Disruption of Nop56/58 homodimerization by alteration of specific amino acids or deletion of the entire coiled-coil domain had no obvious effect upon core protein binding and sRNP assembly. Site-directed mutation of the Nop56/58 homodimerization domain also had no apparent effect upon either box C/D RNP- or C'/D' RNP-guided nucleotide modification. However, deletion of this domain disrupted guided methylation from both RNP complexes. Nuclease probing of the sRNP assembled with Nop56/58 proteins mutated in the coiled-coil domain indicated that while functional complexes were assembled, box C/D and C'/D' RNPs were altered in structure. Collectively, these experiments revealed that the self-dimerization of the Nop56/58 coiled-coil domain is not required for assembly of a functional sRNP, but the coiled-coil domain is important for the establishment of wild-type box C/D and C'/D' RNP structure essential for nucleotide methylation.

Developmentally Programmed Gene Elimination in Euplotes crassus Facilitates a Switch in the Telomerase Catalytic Subunit

The primary function of telomerase is to maintain preexisting telomere tracts. In the ciliate Euplotes crassus, however, telomerase RNP structure and substrate recognition are altered during macronuclear development to facilitate de novo telomere addition. We found that E. crassus harbors three TERT genes encoding the telomerase catalytic subunit that not only vary in their nucleotide and predicted protein sequences, but also in their expression profiles. Expression of EcTERT-1 and -3 correlates with the requirement for telomere maintenance, while that of EcTERT-2 correlates with de novo telomere synthesis. All three genes appear to require ribosomal frameshifting for expression of catalytically active protein. The transcriptionally active form of EcTERT-2 exists only transiently in mated cells and is absent from the vegetative macronucleus. Thus, telomerase expression in Euplotes is controlled by unique regulatory mechanisms that culminate in a developmental switch to a different catalytic subunit with properties suited to de novo telomere addition.

The Clf1p Splicing Factor Promotes Spliceosome Assembly through N-terminal Tetratricopeptide Repeat Contacts

Spliceosome assembly follows a well conserved pathway of subunit addition that includes both small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splicing factors. Clf1p is an unusual splicing factor composed almost entirely of direct repeats of the tetratricopeptide repeat (TPR) protein-binding motif. Here we show that the Clf1p protein resides in at least two multisubunit protein complexes, a small nuclear RNA-free structure similar to what was reported as the Prp19p complex (nineteen complex; NTC) and an RNP structure that contains the U2, U5, and U6 small nuclear RNAs. Thirty Ccf (Clf1p complex factor) proteins have been identified by mass spectroscopy or immune detection as known or suspected components of the yeast spliceosome. Deletion of TPR1 or TPR2 from an epitope-tagged Clf1p protein (i.e. Clf1Delta2-TAP) destabilizes Clf1p complexes assembled in vivo, causing the release of the Cef1p and Prp19p NTC factors and decreased association of the Rse1p, Snu114p, and Hsh155p snRNP proteins. In vitro, temperature inactivation of Clf1Delta2p impairs the prespliceosome to spliceosome transition and prevents Prp19p recruitment to the splicing complex. These and related data support the view that the poly-TPR Clf1p splicing factor promotes the functional integration of the U4/U6.U5 tri-snRNP particle into the U1-, U2-dependent prespliceosome.

Zinc finger domain of Ro60kD autoantigen is essential for binding of Ro52kD and autoantibodies

The Ro60kD polypeptide is associated with both RNA and the Ro52kD protein. The specific protein–RNA and protein–protein interactions are thought to occur through the RNP and zinc-finger secondary structure elements located on the 92–161 and 301–327 regions of Ro60kD protein, respectively. The zinc finger domain of Ro60kD is a good candidate to hold a conformational epitope, because both the binding of zinc and the redox conditions can induce specific conformational changes. In this study, we investigated the presence of antibodies against synthetic peptides corresponding to the zinc finger domain of Ro60kD protein (Zif-1b), to a truncated form possessing zinc-binding regions but lacking the intermediate loop (Zif-2b) and to the intermediate loop (310–319) of the zinc finger domain (Zif-3b). We found that the peptide Zif-1b, corresponding to the native sequence (301–327 aa) of Ro60kD, is recognized by antibodies from the majority (83%) of anti-Ro/La-positive patients with primary Sjogren's syndrome (pSS), in the absence of zinc ions. The same sera failed to react with Zif-1b in the presence of Zn2+ (2.5%). Its truncated form (Zif-2b) did not react against the same sera, while the peptide corresponding to loop 310–319 (Zif-3b) exhibited high reactivity (85%). The presence of zinc ions was necessary for binding of Zif-1b to recombinant Ro52kD, indicating that discrete conformational states of the Ro60kD zinc finger domain are employed in interaction with Ro52kD protein and autoantibodies. The two different states of the zinc finger domain of Ro60kD may reflect the existence of the Ro60kD autoantigen in different redox environments (e.g. in the interior of the cell and cell membrane), where the Cys residues have different capacities to coordinate zinc ions.

A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs.

The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN-Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.

A Common Core RNP Structure Shared between the Small Nucleoar Box C/D RNPs and the Spliceosomal U4 snRNP

The box C/D snoRNAs function in directing 2′-O-methylation and/or as chaperones in the processing of ribosomal RNA. We show here that Snu13p (15.5kD in human), a component of the U4/U6.U5 tri-snRNP, is also associated with the box C/D snoRNAs. Indeed, genetic depletion of Snu13p in yeast leads to a major defect in RNA metabolism. The box C/D motif can be folded into a stem-internal loop-stem structure, almost identical to the 15.5kD binding site in the U4 snRNA. Consistent with this, the box C/D motif binds Snu13p/15.5kD in vitro. The similarities in structure and function observed between the U4 snRNP (chaperone for U6) and the box C/D snoRNPs raises the interesting possibility that these particles may have evolved from a common ancestral RNP.

A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.

We describe the isolation and molecular characterization of seven distinct proteins present in human [U4/U6.U5] tri-snRNPs. These proteins exhibit clear homology to the Sm proteins and are thus denoted LSm (like Sm) proteins. Purified LSm proteins form a heteromer that is stable even in the absence of RNA and exhibits a doughnut shape under the electron microscope, with striking similarity to the Sm core RNP structure. The purified LSm heteromer binds specifically to U6 snRNA, requiring the 3'-terminal U-tract for complex formation. The 3'-end of U6 snRNA was also co-precipitated with LSm proteins after digestion of isolated tri-snRNPs with RNaseT(1). Importantly, the LSm proteins did not bind to the U-rich Sm sites of intact U1, U2, U4 or U5 snRNAs, indicating that they can only interact with a 3'-terminal U-tract. Finally, we show that the LSm proteins facilitate the formation of U4/U6 RNA duplices in vitro, suggesting that the LSm proteins may play a role in U4/U6 snRNP formation.

RNP particles at splice junction sequences on Drosophila chorion transcripts.

HnRNP particles are located at specific sites on nascent transcripts when chromatin is spread for electron microscopic visualization. To determine if the sequences bound by the particles play a role in RNA processing, we have correlated the nascent transcript morphology of Drosophila chorion s36-1 and s38-1 genes with their nucleotide sequences. We find that RNP particles about 25 nm in diameter are at the splice junctions of the introns in these two transcripts. On the more mature chorion transcripts, a single larger (40 nm) particle is occasionally seen in the same vicinity, which probably results from the coalescence of the two smaller particles. This RNP structure may be involved in bringing splice junctions into close proximity and in maintaining this proximity during the bipartite splicing intermediate stage.

Isolation and characterization of nuclear particles containing rapidly labelled hnRNA and snRNA in combination with a distinct set of polypeptides of Mr 74 000 and 72 000

A heterogeneous RNP structure has been isolated from rat liver nuclei by a method previously used for the isolation of 30S RNP complexes carrying heterogeneous RNA (hnRNA) [1]. The RNP sediments in sucrose gradients with s-values of 70–110S. Formaldehyde-fixed preparations band at ρ = 1.40 in isopycnic CsCl gradients. The RNP structure is composed of a heterogeneous population of polypeptides, prominent among which are two proteins with M r 74 000 and 72 000. It contains both rapidly labelled RNA as well as several species of snRNA, as demonstrated by double-labelling experiments and gel electrophonesis. Treatment of rats with α-amanitin leads to a significant decrease in the amount of recovered RNP. In the presence of 0.7 M NaCl the s-value of the complex changes from 70–110S to 40–80S. The RNP structure is stable to mild RNase A or micrococcal nuclease digestion. Transmission electron microscopy reveals the presence of a heterogeneous population of particles with a mean diameter of 300–360 Å. The isolated RNP structure differs completely from the well-known monoparticle or polyparticle hnRNP complexes and from the 30S or smaller snRNP particles but could be similar to or identical with the heterogeneous complex described by Jacob et al. [29].

Ultrastructural analysis of the ribonucleoprotein structure of nascent hnRNA.

Active transcription units are visualized in the electron microscope and the ribonucleoprotein structure of nascent transcripts is analyzed by the technique of RNP fibril mapping. This ultrastructural approach has demonstrated that the nonrandom RNP structure observed is correlated with, and perhaps mediates, a specific RNA processing event.

Phosphorylation of vesicular stomatitis virus proteins as a possible contributing factor in virion uncoating.

The relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of gamma-[32P] ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence of melittin and deoxyadenosine triphosphate, the virion envelope was disrupted based on the accessibility of the internal ribonucleoprotein core (RNP) to the heavy metal stain, uranyl acetate, as determined by electron microscopic observation. The RNP structure was not observed in unphosphorylated virions treated with melittin and uranyl acetate. Phosphorylated virions treated with uranyl acetate subsequently lost the capacity for transcription whereas unphosphorylated virions treated with the stain retained transcriptase activity. These observations suggest that phosphorylation of VSV proteins may contribute to virion uncoating by disrupting the virus envelope.

Correlation of hnRNP structure and nascent transcript cleavage

Using electron microscopy of spread chromatin, we have observed nonnucleolar transcription units from Drosophila melanogaster and Calliphora erythrocephala that display specific cleavage of nascent transcripts. We have quantitatively analyzed 20 of these relatively long transcription units. The primary RNP structure of homologous transcripts is nonrandom with respect to both RNA sequence and the cleavage event. In general, released RNA fragments have a smooth fibrillar RNP morphology (∼50 Å wide) and retained segments have a thicker particulate morphology (∼250 Å diameter). A characteristic secondary structure formation also accompanies cleavage—that is, RNP fibril loops form by association of noncontiguous transcript sequences that correspond to the terminal regions of the segment to be released. RNP particles form at the loop base sequences prior to their association and apparently coalesce upon loop formation. These loops, and thus the released segments, range in length between 1 and 25 kb on the examples we have analyzed. Cleavage of nascent hnRNA transcripts appears to be a fairly common event in these organisms and occurs within 0.3–3 min after transcription of the cleavage site.

Ribonucleoprotein organization of eukaryotic RNA: XV. Different nucleoprotein structures of globin messenger RNA sequences in nuclear and polyribosomal ribonucleoprotein particles

Heterogeneous nuclear RNA and polyribosomal messenger RNA are both complexed with specific sets of proteins in the cell, forming ribonucleoprotein complexes known as hnRNP and mRNP, respectively. In the present investigation, the nucleoprotein structures of globin mRNA sequences in hnRNP and mRNP were probed by digestion with nuclease, under conditions in which RNA-protein rearrangements were shown not to occur. Mild digestion with pancreatic RNAase of a Friend erythroleukemia cell RNP fraction containing both hnRNP and mRNP resulted in a preferential depletion of globin mRNA-homologous sequences, as measured by hybridization of the surviving RNA with globin complementary DNA. Hypersensitivity to nuclease typifies 65% of the globin mRNA-homologous sequences, with the other 35% remaining relatively nuclease-resistant. Removal of polyribosomal mRNP by release with EDTA, followed by re-isolation of hnRNP on a sucrose gradient eliminated the nuclease-hypersensitive class of globin mRNA sequences in favor of the relatively nuclease-resistant class. These results suggest that mRNA sequences are more nuclease-sensitive in polyribosomal mRNP than they are in nuclear hnRNP particles. The implication is that mRNA sequences undergo a significant change in RNP structure at some point during their movement from nucleus to cytoplasm.

Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors.

To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 38,000 mol wt. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with cloned mouse beta-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the beta-globin gene. Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32P-labeled cloned mouse beta-globin DNA reveals the presence in hnRNP of two size classes of beta-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S beta-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S beta-globin mRNA. These results establish, for the first time, that the nuclear transcripts of a specific, well-defined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing.

Ribonucleoprotein structure in nascent hnRNA is nonrandom and sequence-dependent

We have studied the ribonucleoprotein morphology of nascent hnRNA using chromatin spreading methods. This approach allows visualization of multiple transcripts of the same DNA sequence. Thus the RNP structure of hnRNA molecules with the same nucleotide sequence can be compared. We find that RNP particles averaging 240 Å in diameter occur on the majority of hnRNA transcription units, but that RNA fibrils are generally not completely covered with particles. Furthermore, the RNP particle location is nonrandom for the transcripts of a given gene. Particle number and arrangement on RNP fibrils vary from one transcription unit to the next, and are not obviously related to transcription unit size or activity. Analysis of particle location with respect to the 5′ RNA terminus allows RNP particle localization to homologous sequences of less than 500 ribonucleotides on the different fibrils of a single transcription unit. RNP particles assemble very soon after synthesis of the particular sequence. Sites of particle formation are distinct from sites of double-stranded hairpin loops on the RNA fibrils. A differential, or differentially stable, RNP structure with respect to RNA sequence may bear on mechanisms of specific hnRNA processing.