Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly

Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino- or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryote-specific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function.     

Differential assembly of 16S rRNA domains during 30S subunit formation

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.     

Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5′-domain assembly

Ribosomal protein S4 nucleates assembly of the 30S ribosome 5′ and central domains, which is crucial for the survival of cells. Protein S4 changes the structure of its 16S rRNA binding site, passing through a non-native intermediate complex before forming native S4-rRNA contacts. Ensemble FRET was used to measure the thermodynamic stability of non-native and native S4 complexes in the presence of Mg²⁺ ions and other 5′-domain proteins. Equilibrium titrations of Cy3-labeled 5′-domain RNA with Cy5-labeled protein S4 showed that Mg²⁺ ions preferentially stabilize the native S4-rRNA complex. In contrast, ribosomal proteins S20 and S16 act by destabilizing the non-native S4-rRNA complex. The full cooperative switch to the native complex requires S4, S16, and S20 and is achieved to a lesser degree by S4 and S16. The resulting thermodynamic model for assembly of the 30S body illustrates how ribosomal proteins selectively bias the equilibrium between alternative rRNA conformations, increasing the cooperativity of rRNA folding beyond what can be achieved by Mg²⁺ ions alone.     

Assembly of the 5' and 3' minor domains of 16S ribosomal RNA as monitored by tethered probing from ribosomal protein S20

The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5′ domain as well as the 3′ minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5′ domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3′ minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5′ and 3′ minor, are organized relative to S20 at different stages of assembly. The 5′ domain acquires, in a less complex ribonucleoprotein particle than the 3′ minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5′-to-3′ direction assembly model.     

Hemidesmosome Formation Is Initiated by the β4 Integrin Subunit,Requires Complex Formation of β4 and HD1/Plectin,and Involves a Direct Interaction between β4 and the Bullous Pemphigoid Antigen 180

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH₂- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH₂-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.     

Arginine methylation of ribosomal protein S3 affects ribosome assembly

The human ribosomal protein S3 (rpS3), a component of the 40S small subunit in the ribosome, is a known multi-functional protein with roles in DNA repair and apoptosis. We recently found that the arginine residue(s) of rpS3 are methylated by protein arginine methyltransferase 1 (PRMT1). In this paper, we confirmed the arginine methylation of rpS3 protein both in vitro and in vivo. The sites of arginine methylation are located at amino acids 64, 65 and 67. However, mutant rpS3 (3RA), which cannot be methylated at these sites, cannot be transported into the nucleolus and subsequently incorporated into the ribosome. Our results clearly show that arginine methylation of rpS3 plays a critical role in its import into the nucleolus, as well as in small subunit assembly of the ribosome.     

Highly conserved RNA pseudoknots at the gag-pol junction of HIV-1 suggest a novel mechanism of ?1 ribosomal frameshifting

−1 programmed ribosomal frameshifting (PRF) is utilized by many viruses to synthesize their enzymatic (Pol) and structural (Gag) proteins at a defined ratio. For efficient −1 PRF, two cis-acting elements are required: a heptanucleotide frameshift site and a downstream stimulator such as a pseudoknot. We have analyzed the gag-pol junction sequences from 4254 HIV-1 strains. Approximately ninety-five percent of the sequences can form four pseudoknots PK1–PK4 (∼97% contain PK1, PK3, and PK4), covering ∼72 nt including the frameshift site. Some pseudoknots are mutually excluded due to sequence overlap. PK1 and PK3 arrange tandemly. Their stems form a quasi-continuous helix of ∼22 bp. We propose a novel mechanism for possible roles of these pseudoknots. Multiple alternative structures may exist at the gag-pol junction. In most strains, the PK1–PK3 tandem pseudoknots may dominate the structurally heterogeneous pool of RNA due to their greater overall stability. The tandem pseudoknots may function as a breaking system to slow down the ribosome. The ribosome unwinds PK1 and stem 1 of PK3 before it can reach the frameshift site. Then, PK4 can form rapidly because the intact stem 2 of PK3 makes up a large part of the stem 1 of PK4. The newly formed PK4 jams the entrance of the mRNA tunnel. The process then proceeds as in a typical case of −1 PRF. This mechanism incorporates several exquisite new features while still being consistent with the current paradigm of pseudoknot-dependent −1 PRF.     

The sarcin-ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit

The sarcin–ricin loop (SRL) of 23S rRNA in the large ribosomal subunit is a factor-binding site that is essential for GTP-catalyzed steps in translation, but its precise functional role is thus far unknown. Here, we replaced the 15-nucleotide SRL with a GAAA tetraloop and affinity purified the mutant 50S subunits for functional and structural analysis in vitro. The SRL deletion caused defects in elongation-factor-dependent steps of translation and, unexpectedly, loss of EF-Tu-independent A-site tRNA binding. Detailed chemical probing analysis showed disruption of a network of rRNA tertiary interactions that hold together the 23S rRNA elements of the functional core of the 50S subunit, accompanied by loss of ribosomal protein L16. Our results reveal an influence of the SRL on the higher-order structure of the 50S subunit, with implications for its role in translation.     

A single C. elegans PUF protein binds RNA in multiple modes

PUF proteins specifically bind mRNAs to regulate their stability and translation. Here we focus on the RNA-binding specificity of a C. elegans PUF protein, PUF-11. Our findings reveal that PUF-11 binds RNA in multiple modes, in which the protein can accommodate variable spacings between two distinct recognition elements. We propose a structural model in which flexibility in the central region of the protein enables the protein to adopt at least two distinct structures, one of which results in base flipping.     

The C-terminal fragment of ribosomal protein S15 is located in the decoding site of the human ribosome

Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors the mRNA codon at the decoding site and the downstream triplets. The S15 fragment juxtaposed in the human ribosome to mRNA nucleotides +4 to +12 relative to the first nucleotide of the P-site codon was determined. S15 was modified using a set of mRNA analogs containing the triplet UUU/UUC at the 5′ end and a perfluorophenyl azide-carrying uridine at various positions downstream of this triplet. The mRNA analogs were positioned on the ribosome with the use of tRNA^Phe, cognate to the UUU/UUC triplet, targeted to the P site. Modified S15 was isolated from complexes of 80S ribosomes with tRNA^Phe and the mRNA analogs after irradiation with mild UV light and hydrolyzed with cyanogen bromide, cleaving the polypeptide chain after Met residues. Analysis of the modified oligopeptides resulting from hydrolysis demonstrated that the crosslinking site was in C-terminal fragment 111–145 of S15 in all cases, suggesting the involvement of this fragment in the decoding site of the eukaryotic ribosome.     

Cryo-EM structure of SNAP-SNARE assembly in 20S particle

N-ethylmaleimide-sensitive factor (NSF) and α soluble NSF attachment proteins (α-SNAPs) work together within a 20S particle to disassemble and recycle the SNAP receptor (SNARE) complex after intracellular membrane fusion. To understand the disassembly mechanism of the SNARE complex by NSF and α-SNAP, we performed single-particle cryo-electron microscopy analysis of 20S particles and determined the structure of the α-SNAP-SNARE assembly portion at a resolution of 7.35 Å. The structure illustrates that four α-SNAPs wrap around the single left-handed SNARE helical bundle as a right-handed cylindrical assembly within a 20S particle. A conserved hydrophobic patch connecting helices 9 and 10 of each α-SNAP forms a chock protruding into the groove of the SNARE four-helix bundle. Biochemical studies proved that this structural element was critical for SNARE complex disassembly. Our study suggests how four α-SNAPs may coordinate with the NSF to tear the SNARE complex into individual proteins.     

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20_NLS mutant gene and examined polysome profile of cells that had been transfected with the S20_NLS gene. As a result, we observed the formation of recombinant 40S carried S20_NLS but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20_NLS in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20_NLS in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated.     

Insights into the domains required for dimerization and assembly of human alphaB crystallin

Protein pin array technology was used to identify subunit-subunit interaction sites in the small heat shock protein (sHSP) alphaB crystallin. Subunit-subunit interaction sites were defined as consensus sequences that interacted with both human alphaA crystallin and alphaB crystallin. The human alphaB crystallin protein pin array consisted of contiguous and overlapping peptides, eight amino acids in length, immobilized on pins that were in a 96-well ELISA plate format. The interaction of alphaB crystallin peptides with physiological partner proteins, alphaA crystallin and alphaB crystallin, was detected using antibodies and recorded using spectrophotometric absorbance. Five peptide sequences including 37LFPTSTSLSPFYLRPPSF54 in the N terminus, 75FSVNLDVK82)(beta3), 131LTITSSLS138 (beta8) and 141GVLTVNGP148 (beta9) that form beta strands in the conserved alpha crystallin core domain, and 155PERTIPITREEK166 in the C-terminal extension were identified as subunit-subunit interaction sites in human alphaB crystallin using the novel protein pin array assay. The subunit-subunit interaction sites were mapped to a three-dimensional (3D) homology model of wild-type human alphaB crystallin that was based on the crystal structure of wheat sHSP16.9 and Methanococcus jannaschi sHSP16.5 (Mj sHSP16.5). The subunit-subunit interaction sites identified and mapped onto the homology model were solvent-exposed and had variable secondary structures ranging from beta strands to random coils and short alpha helices. The subunit-subunit interaction sites formed a pattern of hydrophobic patches on the 3D surface of human alphaB crystallin.     

Identification of an Escherichia coli S1-like protein in the spinach chloroplast ribosome

Antibodies directed against E. coli ribosomal protein S1 were used in immunoblotting assays to search for an S1-like protein in the ribosome of spinach chloroplast. An immunological cross-reaction was reproducibly detected on the blots and inhibition experiments have demonstrated its specificity. The chloroplastic ribosomal protein which has epitopes common to antigenic determinants of the E. coli protein S1 was identified as being protein S2/S3.     

Enzyme dimension of the ribosomal protein S4 across plant and animal kingdoms

Background

The protein S4 of the smaller ribosomal subunit is centrally important for its anchorage role in ribosome assembly and rRNA binding. Eubacterial S4 also facilitates synthesis of rRNA, and restrains translation of ribosomal proteins of the same polycistronic mRNA. Eukaryotic S4 has no homolog in eubacterial kingdom, nor are such extraribosomal functions of S4 known in plants and animals even as genetic evidence suggests that deficiency of S4X isoform in 46,XX human females may produce Turner syndrome (45,XO).

Methods

Recombinant human S4X and rice S4 were used to determine their enzymatic action in the cleavage of synthetic peptide substrates and natural proteins. We also studied autoproteolysis of the recombinant S4 proteins, and examined the growth and proliferation of S4-transfected human embryonic kidney cells.

Results

Extraribosomal enzyme nature of eukaryotic S4 is described. Both human S4X and rice S4 are cysteine proteases capable of hydrolyzing a wide spectrum of peptides and natural proteins of diverse origin. Whereas rice S4 also cleaves the -XXXD↓- consensus sequence assumed to be specific for caspase-9 and granzyme B, human S4 does not. Curiously, both human and rice S4 show multiple-site autoproteolysis leading to self-annihilation. Overexpression of human S4 blocks the growth and proliferation of transfected embryonic kidney cells, presumably due to the extraribosomal enzyme trait reported.

Conclusions

The S4 proteins of humans and rice, prototypes of eukaryota, are non-specific cysteine proteases in the extraribosomal milieu.

General significance

The enzyme nature of S4 is relevant toward understanding not only the origin of ribosomal proteins, but also processes in cell biology and diseases.     

Mapping the essential structures of human ribosomal protein L7 for nuclear entry, ribosome assembly and function

Human large subunit protein L7 carries multiple nuclear localization signals (NLS) in its structure: there are three monobasic partite NLSs at the NH2-region of the first 54 amino acid residues and a bipartite in the middle section at position of 156-167. The C-region of the last 50 amino acid residues displays membrane binding nature, and might involve in forming a nuclear microbody for pre-nucleolar ribosome assembly. The middle section covers 144 amino acid residues which are essential for the structure and function of ribosome. This is evident from findings that truncated L7 without the NH2-region or the C-region, or missing both regions, is capable of reaching nucleolus and incorporating in ribosome, however, only ribosomes bearing truncated L7 without the NH2-region is capable of engaging in polysome formation. Combining with the phylogenic findings from homologous sequence alignment, the NH2-region of L7, besides being as a eukaryotic expansion segment, can be excluded from building a functional eukaryotic ribosome.     

Analysis of the interactome of ribosomal protein S19 mutants

Diamond–Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one‐fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild‐type protein one, using the same functional proteomic approach that we employed to characterize RPS19 interactome. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. Results presented in this paper highlight the striking differences between the interactomes of wild‐type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control. The data have been deposited to the ProteomeXchange with identifier PXD000640 ( http://proteomecentral.proteomexchange.org/dataset/PXD000640 ).     

The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5'-translational operator mRNA

The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds a purine-rich three-helix junction motif in the central domain of 16S ribosomal RNA (rRNA) as well as a translational operator located in the 5'-untranslated region (5'-UTR) of its cognate messenger RNA (mRNA). An in-frame fusion between the 5'-UTR of the BS15 gene and beta-galactosidase (lacZ) was prepared, and tested for BS15-dependent translational repression of lacZ activity in Escherichia coli. The presence of BS15 in trans represses lacZ activity 24-fold. A series of detailed point mutations in BS15 were tested for their effects upon translational repression of lacZ activity. These point mutations demonstrated that the 5'-UTR-BS15 binding interface utilizes many of the same conserved amino acid residues implicated in the binding of BS15 to 16S rRNA. The data demonstrate that the S15 protein can bind to an RNA target motif based primarily upon appropriate minor groove and sugar-phosphate backbone contacts, irrespective of the specific RNA sequence.     

Phactr4: A new integrin modulator required for directional migration of enteric neural crest cells

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.