A dynamic scaffold of pre-snoRNP factors facilitates human box C/D snoRNP assembly

The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. The core box C/D proteins are restructured during human U3 box C/D snoRNP biogenesis; however, the molecular basis of this is unclear. Here we show that the U8 snoRNP is also restructured, suggesting that this may occur with all box C/D snoRNPs. We have characterized four novel human biogenesis factors (BCD1, NOP17, NUFIP, and TAF9) which, along with the ATPases TIP48 and TIP49, are likely to be involved in the formation of the pre-snoRNP. We have analyzed the in vitro protein-protein interactions between the assembly factors and core box C/D proteins. Surprisingly, this revealed few interactions between the individual core box C/D proteins. However, the novel biogenesis factors and TIP48 and TIP49 interacted with one or more of the core box C/D proteins, implying that they mediate the assembly of the pre-snoRNP. Consistent with this, we show that NUFIP bridges interactions between the core box C/D proteins in a partially reconstituted pre-snoRNP. Restructuring of the core complex probably reflects the conversion of the pre-snoRNP, where core protein-protein interactions are maintained by the bridging biogenesis factors, to the mature snoRNP.     

Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP

Methylation of the ribose 2'-hydroxyl, the most widespread modification of ribosomal and splicesomal RNAs, is guided by the box C/D class of small nucleolar RNAs (snoRNAs). Box C/D small nucleolar ribonucleoproteins (snoRNPs) contain four core proteins: fibrillarin, Nop56, Nop58 and 15.5 kDa. We constructed U25 snoRNAs containing a single photoactivatable 4-thiouridine at each U position within the conserved box C/D and C'/D' motifs. Proteins assembled on the snoRNA after injection into Xenopus oocyte nuclei were identified by cross-linking, and reconstituted particles characterized by functional rescue and mutational analyses. Our data argue that box C/D snoRNPs are asymmetric, with the C' box contacting Nop56 and fibrillarin, the C box interacting with Nop58, and the D and D' boxes contacting fibrillarin. No cross-link to 15.5 kDa was detected; its binding is disrupted by 4-thiouridine substitution in position 1 of the C box. Repositioning the guide sequence of U25 upstream of box D instead of D' revealed that both C/D motifs have the potential to function as guide centers, but, surprisingly, there was no alteration in protein cross-linking.     

Involvement of nuclear import and export factors in U8 box C/D snoRNP biogenesis

Box C/D snoRNPs, factors essential for ribosome biogenesis, are proposed to be assembled in the nucleoplasm before localizing to the nucleolus. However, recent work demonstrated the involvement of nuclear export factors in this process, suggesting that export may take place. Here we show that there are distinct distributions of U8 pre-snoRNAs and pre-snoRNP complexes in HeLa cell nuclear and cytoplasmic extracts. We observed differential association of nuclear export (PHAX, CRM1, and Ran) factors with complexes in the two extracts, consistent with nucleocytoplasmic transport. Furthermore, we show that the U8 pre-snoRNA in one of the cytoplasmic complexes contains an m3G cap and is associated with the nuclear import factor Snurportin1. Using RNA interference, we show that loss of either PHAX or Snurportin1 results in the incorrect localization of the U8 snoRNA. Our data therefore show that nuclear export and import factors are directly involved in U8 box C/D snoRNP biogenesis. The distinct distribution of U8 pre-snoRNP complexes between the two cellular compartments together with the association of both nuclear import and export factors with the precursor complex suggests that the mammalian U8 snoRNP is exported during biogenesis.     

Control of box C/D snoRNP assembly by N6‐methylation of adenine

N⁶‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N⁶‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N⁶‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N⁶mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.     

In vitro assembly of U1 snRNPs.

An efficient system for the in vitro assembly of U1 snRNPs is described. RNA-protein interactions in a series of U1 snRNA mutants assembled both in vivo and in vitro were studied in order to verify the accuracy of the system. Two discrete protein binding sites are defined by immunoprecipitation with antibodies against different protein components of the U1 snRNP and a newly developed protein sequestering assay. The U1 snRNP-specific proteins 70K and A require only the 5'-most stem-loop structure of U1 snRNA for binding, the common U snRNP proteins require the conserved Sm binding site (AUnG). Interactions between these two groups of proteins are detected. These results are combined to derive a model of the U1 snRNP structure. The potential use of the in vitro system in the functional analysis of U1 snRNP proteins is discussed.     

A spliceosomal intron binding protein, IBP160, links position-dependent assembly of intron-encoded box C/D snoRNP to pre-mRNA splicing

Pre-mRNA splicing in vertebrates is molecularly linked to other processes. We previously reported that splicing is required for efficient assembly of intron-encoded box C/D small nucleolar ribonucleoprotein (snoRNP). In the spliceosomal C1 complex, snoRNP proteins efficiently assemble onto snoRNA sequences if they are located about 50 nt upstream of the intron branchpoint. Here, we identify the splicing factor responsible for coupling snoRNP assembly to intron excision. Intron binding protein (IBP) 160, a helicase-like protein previously detected in the spliceosomal C1 complex, binds the pre-mRNA in a sequence-independent manner, contacting nucleotides 33-40 upstream of the intron branch site, regardless of whether a snoRNA is present. Depletion of IBP160 abrogates snoRNP assembly in vitro. IBP160 binding directly to a snoRNA located too close to the intron branch site interferes with snoRNP assembly. Thus, IBP160 is the key factor linking snoRNP biogenesis and perhaps other postsplicing events to pre-mRNA splicing.     

Protein Hit1, a novel box C/D snoRNP assembly factor,controls cellular concentration of the scaffolding protein Rsa1 by direct interaction

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82–R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3′-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p_317–352–Hit1p_70–164 complex reveals a novel mode of protein–protein association explaining the strong stability of the Rsa1p–Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p–Rsa1p–Hit1p heterotrimer.     

Comparative analysis of the 15.5kD box C/D snoRNP core protein in the primitive eukaryote Giardia lamblia reveals unique structural and functional features

Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 ?. The Giardia 15.5kD protein exhibits the typical α-β-α sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.     

The spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of the eukaryotic box C/D snoRNP nucleotide modification complex

Box C/D ribonucleoprotein particles guide the 2'-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C'/D' motifs in the box C/D RNA. The C/D and C'/D' RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2'-O-methylation when the C'/D' motif was either mutated or ablated. In contrast, the C'/D' RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C'/D' RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C'/D' motifs. Therefore, the spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of both archaeal and eukaryotic box C/D RNP complexes.     

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

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.     

Nutritional status modulates box C/D snoRNP biogenesis by regulated subcellular relocalization of the R2TP complex

Background

Box C/D snoRNPs, which are typically composed of box C/D snoRNA and the four core protein components Nop1, Nop56, Nop58, and Snu13, play an essential role in the modification and processing of pre-ribosomal RNA. The highly conserved R2TP complex, comprising the proteins Rvb1, Rvb2, Tah1, and Pih1, has been shown to be required for box C/D snoRNP biogenesis and assembly; however, the molecular basis of R2TP chaperone-like activity is not yet known.

Results

Here, we describe an unexpected finding in which the activity of the R2TP complex is required for Nop58 protein stability and is controlled by the dynamic subcellular redistribution of the complex in response to growth conditions and nutrient availability. In growing cells, the complex localizes to the nucleus and interacts with box C/D snoRNPs. This interaction is significantly reduced in poorly growing cells as R2TP predominantly relocalizes to the cytoplasm. The R2TP-snoRNP interaction is mainly mediated by Pih1.

Conclusions

The R2TP complex exerts a novel regulation on box C/D snoRNP biogenesis that affects their assembly and consequently pre-rRNA maturation in response to different growth conditions.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0404-4) contains supplementary material, which is available to authorized users.     

In vitro assembly and disassembly of 14-nm filament from Tetrahymena pyriformis. The protein component of 14-nm filament is a 49,000-Dalton protein

Conditions for in vitro assembly and disassembly of Tetrahymena 14-nm filaments were investigated electron-microscopically by using a crude extract of acetone powder of the cells. The assembly conditions established are: incubation of a protein sample (2 mg/ml) in 5 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.6) containing 0.1 mM N alpha-tosyl-L-lysyl-chloromethane hydrochloride (TLCK), 50 mM KCl, 0.6 mM ATP, and 1.2 mM CaCl2 at 30 degrees C for 30 min. The disassembly conditions established are: dialysis of the 14-nm filament suspension (3 mg protein/ml) against Tris-acetate buffer (pH 8.2) containing 5 mM 2-mercaptoethanol, 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and 0.05 mM TLCK at 4 degrees C for 24 h. The assembly and disassembly were repeatable, and resulted in the exclusive retention of the 49,000-dalton protein. This clearly shows that the previously reported protein component (38,000-dalton protein : FFP-38) of the 14-nm filament is incorrect and the actual component is indeed a 49,000-dalton protein. The present research also showed that the Tetrahymena 14-nm filament bore a strong resemblance to 'intermediate filaments' of mammalian cells with respect to molecular weight, amino-acid composition of the protein component, and size and conditions for assembly and disassembly of the filament.     

The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro.

Stable association of the eight common Sm proteins with U1, U2, U4 or U5 snRNA to produce a spliceosomal snRNP core structure is required for snRNP biogenesis, including cap hypermethylation and nuclear transport. Here, the assembly of snRNP core particles was investigated in vitro using both native HeLa and in vitro generated Sm proteins. Several RNA-free, heteromeric protein complexes were identified, including E.F.G, B/B'.D3 and D1.D2.E.F.G. While the E.F.G complex alone did not stably bind to U1 snRNA, these proteins together with D1 and D2 were necessary and sufficient to form a stable U1 snRNP subcore particle. The subcore could be chased into a core particle by the subsequent addition of the B/B'.D3 protein complex even in the presence of free competitor U1 snRNA. Trimethylation of U1 snRNA's 5' cap, while not observed for the subcore, occurred in the stepwise-assembled U1 snRNP core particle, providing evidence for the involvement of the B/B' and D3 proteins in the hypermethylation reaction. Taken together, these results suggest that the various protein heterooligomers, as well as the snRNP subcore particle, are functional intermediates in the snRNP core assembly pathway.     

Hepatitis C virus core protein binds to a DEAD box RNA helicase.

Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation.     

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2'',5''-phosphodiester bond (RNA branch) with the 5'' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.     

In vitro binding of cattle PstI SINE with a 33-kDa nuclear protein.

A PstI family of SINEs (short interspersed elements) has been identified in some of the members of the family Bovidae, for example, cattle, buffalo and goat. In vitro DNA-protein interactions were studied to provide a better understanding of the function of these SINEs in the genome. Use of one such cattle PstI interspersed repeat sequence, as a probe in gel retardation assays, has lead to the identification of a repeat DNA-binding factor PIRBP (PstI interspersed repeat binding protein) from cattle liver nuclear extract. Southwestern analysis with liver nuclear extracts from cattle, goat, and buffalo revealed the presence of a PIRBP-like nuclear factor in all three species belonging to the family Bovidae. Deletion analysis localized the PIRBP binding site to an 80-bp (337-417 bp) region within the cattle PstI sequence. UV crosslinking and Southwestern analyses clearly indicated that PIRBP is a singular, small polypeptide of 33-kDa molecular mass. Homology search of the nucleic acids database revealed that the cattle PstI sequence was associated with many different genes of the family Bovidae, either in the 5' flanking region, 5' locus activating region, 3' UTR or in intervening sequences. The binding of the cattle PstI SINE by PIRBP and its association with the regulatory regions of the genes suggests that it plays an important role in the bovine genome.     

Dhr1p, a putative DEAH-box RNA helicase, is associated with the box C+D snoRNP U3

Putative RNA helicases are involved in most aspects of gene expression. All previously characterized members of the DEAH-box family of putative RNA helicases are involved in pre-mRNA splicing. Here we report the analysis of two novel DEAH-box RNA helicases, Dhr1p and Dhr2p, that were found to be predominantly nucleolar. Both genes are essential for viability, and MET-regulated alleles were therefore created. Depletion of Dhr1p or Dhr2p had no detectable effect on pre-mRNA splicing in vivo or in vitro. Both Dhr1p and Dhr2p were, however, required for 18S rRNA synthesis. Depletion of Dhr2p inhibited pre-rRNA cleavage at sites A(0), A(1), and A(2), while Dhr1p depletion inhibited cleavage at sites A(1) and A(2). No coprecipitation of snoRNAs was detected with ProtA-Dhr2p, but Dhr1p-ProtA was stably associated with the U3 snoRNA. Depletion of Dhr1p inhibited processing steps that require base pairing of U3 to the 5' end of the 18S rRNA. We speculate that Dhr1p is targeted to the preribosomal particles by the U3-18S rRNA interaction and is required for the structural reorganization of the rRNA during formation of the central pseudoknot.