Direct binding of the Alu binding protein dimer SRP9/14 to 40S ribosomal subunits promotes stress granule formation and is regulated by Alu RNA.

Stress granules (SGs) are formed in response to stress, contain mRNAs, 40S ribosomal subunits, initiation factors, RNA-binding and signaling proteins, and promote cell survival. Our study describes a novel function of the protein heterodimer SRP9/14 and Alu RNA in SG formation and disassembly. In human cells, SRP9/14 exists assembled into SRP, bound to Alu RNA and as a free protein. SRP9/14, but not SRP, localizes to SGs following arsenite or hippuristanol treatment. Depletion of the protein decreases SG size and the number of SG-positive cells. Localization and function of SRP9/14 in SGs depend primarily on its ability to bind directly to the 40S subunit. Binding of SRP9/14 to 40S and Alu RNA is mutually exclusive indicating that the protein alone is bound to 40S in SGs and that Alu RNA might competitively regulate 40S binding. Indeed, by changing the effective Alu RNA concentration in the cell or by expressing an Alu RNA binding-defective protein we were able to influence SG formation and disassembly. Our findings suggest a model in which SRP9/14 binding to 40S promotes SG formation whereas the increase in cytoplasmic Alu RNA following stress promotes disassembly of SGs by disengaging SRP9/14 from 40S.

Useful 'junk': Alu RNAs in the human transcriptome.

Alu elements are the most abundant repetitive elements in the human genome; they have amplified by retrotransposition to reach the present number of more than one million copies. Alu elements can be transcribed in two different ways, by two independent polymerases. 'Free Alu RNAs' are transcribed by Pol III from their own promoter, while 'embedded Alu RNAs' are transcribed by Pol II as part of protein- and non-protein-coding RNAs. Recent studies have demonstrated that both free and embedded Alu RNAs play a major role in post transcriptional regulation of gene expression, for example by affecting protein translation, alternative splicing and mRNA stability. These discoveries illustrate how a part of the 'junk DNA' content of the human genome has been recruited to important functions in regulation of gene expression.

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle.

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5' domain. This creates the binding site for the Alu RNA 3' domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3' domain folding back by 180 degrees onto the 5' domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.

Structure and assembly of the Alu domain of the mammalian signal recognition particle.

The Alu domain of the mammalian signal recognition particle (SRP) comprises the heterodimer of proteins SRP9 and SRP14 bound to the 5' and 3' terminal sequences of SRP RNA. It retards the ribosomal elongation of signal-peptide-containing proteins before their engagement with the translocation machinery in the endoplasmic reticulum. Here we report two crystal structures of the heterodimer SRP9/14 bound either to the 5' domain or to a construct containing both 5' and 3' domains. We present a model of the complete Alu domain that is consistent with extensive biochemical data. SRP9/14 binds strongly to the conserved core of the 5' domain, which forms a U-turn connecting two helical stacks. Reversible docking of the more weakly bound 3' domain might be functionally important in the mechanism of translational regulation. The Alu domain structure is probably conserved in other cytoplasmic ribonucleoprotein particles and retroposition intermediates containing SRP9/14-bound RNAs transcribed from Alu repeats or related elements in genomic DNA.

The Alu domain homolog of the yeast signal recognition particle consists of an Srp14p homodimer and a yeast-specific RNA structure.

The mammalian Alu domain of the signal recognition particle (SRP) consists of a heterodimeric protein SRP9/14 and the Alu portion of 7SL RNA and comprises the elongation arrest function of the particle. To define the domain in Saccharomyces cerevisiae SRP that is homologous to the mammalian Alu domain [Alu domain homolog in yeast (Adhy)], we examined the assembly of a yeast protein homologous to mammalian SRP14 (Srp14p) and scR1 RNA. Srp14p binds as a homodimeric complex to the 5' sequences of scR1 RNA. Its minimal binding site consists of 99 nt. (Adhy RNA), comprising a short hairpin structure followed by an extended stem. As in mammalian SRP9/14, the motif UGUAAU present in most SRP RNAs is part of the Srp14p binding sites as shown by footprint and mutagenesis studies. In addition, certain basic amino acid residues conserved between mammalian SRP14 and Srp14p are essential for RNA binding in both proteins. These findings confirm the common ancestry of the yeast and the mammalian components and indicate that Srp14p together with Adhy RNA represents the Alu domain homolog in yeast SRP that may comprise its elongation arrest function. Despite the similarities, Srp14p selectively recognizes only scR1 RNA, revealing substantial changes in RNA-protein recognition as well as in the overall structure of the complex. The alignment of the three yeast SRP RNAs known to date suggests a common structure for the putative elongation arrest domain of all three organisms.

New insights into signal recognition and elongation arrest activities of the signal recognition particle.

The signal recognition particle (SRP), a ubiquitous cytoplasmic ribonucleoprotein particle, plays an essential role in promoting co-translational translocation of proteins into the endoplasmic reticulum. Here, we summarise recent progress made in the understanding of two essential SRP functions: the signal recognition function, which ensures the specificity, and the elongation arrest function, which increases the efficiency of translocation. Our discussion is based on functional data as well as on atomic structure information, both of which also support the notion that SRP is a very ancient particle closely related to ribosomes. Based on the significant increase of knowledge that has been accumulating on the structure of elongation factors and on their interactions with the ribosome, we speculate about a possible mechanism of the elongation arrest function.

Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14.

We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRPphi14-9. Recombinant SRPphi14-9 is a fusion protein containing on a single polypeptide chain the sequences of both the SRP14 and SRP9 proteins that are part of the Alu domain of the signal recognition particle (SRP). SRPphi14-9 has been shown to bind to the 7SL RNA of SRP and it confers elongation arrest activity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3' ends were produced in vitro using ribozyme technology and tested for specific SRPphi14-9 binding in a quantitative equilibrium competition assay. This enabled identification of an Alu RNA of 86 nt (SA86) that competes efficiently with 7SL RNA for SRPphi14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two stem-loops that are connected by a highly conserved bulge and, in addition, a part of the central adaptor stem that contains the sequence at the very 3' end of 7SL RNA. Circularly permuted variants of SA86 competed only if the 5' and 3' ends were joined with an extended linker of four nucleotides. SA86 can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for specific recognition by SRPphi14-9. These results suggest that Alu RNA identity is determined by a characteristic tertiary structure, which might consist of two flexibly linked domains.

The crystal structure of the signal recognition particle Alu RNA binding heterodimer, SRP9/14.

The mammalian signal recognition particle (SRP) is an 11S cytoplasmic ribonucleoprotein that plays an essential role in protein sorting. SRP recognizes the signal sequence of the nascent polypeptide chain emerging from the ribosome, and targets the ribosome-nascent chain-SRP complex to the rough endoplasmic reticulum. The SRP consists of six polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) and a single 300 nucleotide RNA molecule. SRP9 and SRP14 proteins form a heterodimer that binds to the Alu domain of SRP RNA which is responsible for translation arrest. We report the first crystal structure of a mammalian SRP protein, that of the mouse SRP9/14 heterodimer, determined at 2.5 A resolution. SRP9 and SRP14 are found to be structurally homologous, containing the same alpha-beta-beta-beta-alpha fold. This we designate the Alu binding module (Alu bm), an additional member of the family of small alpha/beta RNA binding domains. The heterodimer has pseudo 2-fold symmetry and is saddle like, comprising a strongly curved six-stranded amphipathic beta-sheet with the four helices packed on the convex side and the exposed concave surface being lined with positively charged residues.

Mutational analysis of the protein subunits of the signal recognition particle Alu-domain.

Two polypeptides of the murine signal recognition particle (SRP), SRP9 and SRP14, bind exclusively as a heterodimer to SRP RNA and their presence is required for elongation arrest activity of the particle. SRP9/14 also constitute a subunit of small cytoplasmic Alu RNPs. To identify RNA-binding determinants, we assayed the dimerization and RNA-binding capacities of altered proteins in vitro. Despite the structural homology of the two proteins, their requirements for dimerization differ substantially. In SRP9, an internal fragment of 43 amino acids is sufficient to allow dimer formation, whereas in SRP14 only few changes, such as removing an internal loop region, are tolerated without affecting its dimerization activity. The dimerization defect of the SRP14 proteins is most likely explained by a reduced stability or ability to fold of the proteins. Interestingly, SRP RNA can engage certain dimerization-defective SRP14 proteins into stable complexes, suggesting that low-affinity interactions between the RNA and SRP14 may help to overcome the folding defect or the reduced stability of the proteins. We identified two regions, one in each protein, that are essential for RNA-binding. In SRP9, acidic amino acid residues in the N-terminal alpha-helix and the adjacent loop and, in SRP14, a flexible internal loop region are critical for RNA-binding. In the heterodimer, the two regions are located in close proximity, consistent with the RNA-binding region being formed by both proteins.

A truncation in the 14 kDa protein of the signal recognition particle leads to tertiary structure changes in the RNA and abolishes the elongation arrest activity of the particle.

The signal recognition particle (SRP) provides the molecular link between synthesis of polypeptides and their concomitant translocation into the endoplasmic reticulum. During targeting, SRP arrests or delays elongation of the nascent chain, thereby presumably ensuring a high translocation efficiency. Components of the Alu domain, SRP9/14 and the Alu sequences of SRP RNA, have been suggested to play a role in the elongation arrest function of SRP. We generated a truncated SRP14 protein, SRP14-20C, which forms, together with SRP9, a stable complex with SRP RNA. However, particles reconstituted with SRP9/14-20C, RC(9/14-20C), completely lack elongation arrest activity. RC(9/14-20C) particles have intact signal recognition, targeting and ribosome binding activities. SRP9/14-20C therefore only impairs interactions with the ribosome that are required to effect elongation arrest. This result provides evidence that direct interactions between the Alu domain components and the ribosome are required for this function. Furthermore, SRP9/14-20C binding to SRP RNA results in tertiary structure changes in the RNA. Our results strongly indicate that these changes account for the negative effect of SRP14 truncation on elongation arrest, thus revealing a critical role of the RNA in this function.

The SRP9/14 subunit of the human signal recognition particle binds to a variety of Alu-like RNAs and with higher affinity than its mouse homolog.

The heterodimeric subunit, SRP9/14, of the signal recognition particle (SRP) has previously been found to bind to scAlu and scB1 RNAs in vitro and to exist in large excess over SRP in anthropoid cells. Here we show that human and mouse SRP9/14 bind with high affinities to other Alu-like RNAs of different evolutionary ages including the neuron-specific BC200 RNA. The relative dissociation constants of the different RNA-protein complexes are inversely proportional to the evolutionary distance between the Alu RNA species and 7SL RNA. In addition, the human SRP9/14 binds with higher affinity than mouse SRP9/14 to all RNAs analyzed and this difference is not explained by the additional C-terminal domain present in the anthropoid SRP14. The conservation of high affinity interactions between SRP9/14 and Alu-like RNAs strongly indicates that these Alu-like RNPs exist in vivo and that they have cellular functions. The observation that human SRP9/14 binds better than its mouse counterpart to distantly related Alu RNAs, such as recently transposed elements, suggests that the anthropoid-specific excess of SRP9/14 may have a role in controlling Alu amplification rather than in compensating a defect in SRP assembly and functions.

[Local infiltration of epinephrine and bupivacaine before tonsillectomy]. / Zur lokalen Infiltration von Epinephrin und Bupivacain vor Tonsillektomie.

The effects of peritonsillar injections of epinephrine and local anesthetics before tonsillectomy on blood loss and postoperative pain were evaluated in a prospective, randomized double-blind trial on 103 children. Patients were randomly assigned into one of three groups: controls given injections of 0.9% NaCl (n = 34), patients injected with 0.4 ml/kg (1:200,000) epinephrine combined with 0.25% bupivacaine (n = 33) and patients given only 1:200,000 epinephrine (n = 36). All injections and operations were performed by the same surgeon (KS). Blood loss was calculated by weighing all blood aspirated perioperatively and swabs used during surgery. Postoperative pain was assessed at regular intervals by using three methods: (1) use of a visual analogue scale by parents and nurses to estimate pain; (2) postoperative need for nalbuphine as analgesic; (3) the Hannallah-Broadman semi-objective pain score (including crying, anxiety, restlessness, and changes in blood pressure). The mean blood loss in the control group (given NaCl) was 132 g, which was significantly increased when compared with the epinephrine/bupivacaine group (85 g) and the group treated with only epinephrine (90 g). However, analysis of the postoperative pain scores did not reveal any significant differences among groups. These findings indicate that the peritonsillar injection of bupivacaine does not decrease postoperative pain, but peritonsillar injections of epinephrine will significantly reduce blood loss during tonsillectomy.

The signal recognition particle and related small cytoplasmic ribonucleoprotein particles.

Recently, a number of novel small cytoplasmic ribonucleoprotein particles have been identified that comprise RNA and protein subunits related to the signal recognition particle (SRP). Here we discuss the latest results on the structure and functions of SRP together with the structures and putative functions of the novel SRP-related ribonucleoprotein particles.

Crystallization and preliminary crystallographic analysis of the signal recognition particle SRPphi14-9 fusion protein.

The SRPphi14-9 fusion protein, which can functionally replace the SRP9/14 heterodimer in the mammalian signal recognition particle (SRP), has been crystallized using the vapor diffusion method. Four different crystal forms were grown. SRPphi14-9 form IV crystals belong to the space group P4(1)22/ P4(3)22 with cell parameters a = b = 69.7 Angstroms, c = 95.7 Angstroms, alpha = beta = gamma = 90 degrees. A complete data set to 2.8 Angstroms resolution with an Rsym on intensities of 7.0% was collected on a single flash-frozen crystal.

Crystallization and preliminary X-ray analysis of the 9 kDa protein of the mouse signal recognition particle and the selenomethionyl-SRP9.

Two different crystal forms of the 9 kDa protein of the signal recognition particle (SRP9) have been prepared by the hanging drop vapor diffusion technique using 28% (w/v) PEG8000 or 28% saturated ammonium sulphate as precipitant. The crystals are hexagonal bipyramids with average dimensions of 0.2 X 0.1 X 0.1 mm(3) and they diffract to a resolution of 2.3 Angstroms. They belong to the space groups P6(2)22/P6(4)22 or P3(1)21/P3(2)21 with cell dimensions a = b = 63.0 Angstroms, and c = 111.5 Angstroms. Crystals have also been grown from the selenomethionyl protein and multiwavelength data sets have been collected.

A two-step recognition of signal sequences determines the translocation efficiency of proteins.

The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery.

The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs.

The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism.

The heterodimeric subunit SRP9/14 of the signal recognition particle functions as permuted single polypeptide chain.

The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP). The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle. SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA. We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide. We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure. Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio. In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay. Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities. The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro.

Binding sites of the 9- and 14-kilodalton heterodimeric protein subunit of the signal recognition particle (SRP) are contained exclusively in the Alu domain of SRP RNA and contain a sequence motif that is conserved in evolution.

The mammalian signal recognition particle (SRP) is a small cytoplasmic ribonucleoprotein required for the cotranslational targeting of secretory proteins to the endoplasmic reticulum membrane. The heterodimeric protein subunit SRP9/14 was previously shown to be essential for SRP to cause pausing in the elongation of secretory protein translation. RNase protection and filter binding experiments have shown that binding of SRP9/14 to SRP RNA depends solely on sequences located in a domain of SRP RNA that is strongly homologous to the Alu family of repetitive DNA sequences. In addition, the use of hydroxyl radicals, as RNA-cleaving reagents, has revealed four distinct regions in this domain that are in close contact with SRP9/14. Surprisingly, the nucleotide sequence in one of these contact sites, predicted to be mostly single stranded, was found to be extremely conserved in SRP RNAs of evolutionarily distant organisms ranging from eubacteria and archaebacteria to yeasts and higher eucaryotic cells. This finding suggests that SRP9/14 homologs may also exist in these organisms, where they possibly contribute to the regulation of protein synthesis similar to that observed for mammalian SRP in vitro.