RN7SL1 may be translated under oncogenic conditions.

RN7SL1 (RNA component of signal recognition particle 7SL1), a component of the signal recognition particle, is a non-coding RNA possessing a small ORF (smORF). However, whether it is translated into peptides is unknown. Here, we generated the RN7SL1-Green Fluorescent Protein (GFP) gene, in which the smORF of RN7SL1 was replaced by GFP, introduced it into 293T cells, and observed cells emitting GFP fluorescence. Furthermore, RNA-seq of GFP-positive cells revealed that they were in an oncogenic state, suggesting that RN7SL1 smORF may be translated under special conditions.

Loss of Preproinsulin Interaction with Signal Recognition Particle Activates Protein Quality Control, Decreasing mRNA Stability.

Many insulin gene variants alter the protein sequence and result in monogenic diabetes due to insulin insufficiency. However, the molecular mechanisms of various disease-causing mutations are unknown. Insulin is synthesized as preproinsulin containing a signal peptide (SP). SPs of secreted proteins are recognized by the signal recognition particle (SRP) or by another factor in a SRP-independent pathway. If preproinsulin uses SRP-dependent or independent pathways is still debatable. We demonstrate by the use of site-specific photocrosslinking that the SRP subunit, SRP54, interacts with the preproinsulin SP. Moreover, SRP54 depletion leads to the decrease of insulin mRNA and protein expression, supporting the involvement of the RAPP protein quality control in insulin biogenesis. RAPP regulates the quality of secretory proteins through degradation of their mRNA. We tested five disease-causing mutations in the preproinsulin SP on recognition by SRP and on their effects on mRNA and protein levels. We demonstrate that the effects of mutations are associated with their position in the SP and their severity. The data support diverse molecular mechanisms involved in the pathogenesis of these mutations. We show for the first time the involvement of the RAPP protein quality control pathway in insulin biogenesis that is implicated in the development of neonatal diabetes caused by the Leu13Arg mutation.

The P124A mutation of SRP14 alters its migration on SDS-PAGE without impacting its function.

SRP14 is a crucial protein subunit of the signal recognition particle (SRP), a ribonucleoprotein complex essential for co-translational translocation to the endoplasmic reticulum. During our investigation of SRP14 expression across diverse cell lines, we observe variations in its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with some cells exhibiting slower migration and others migrating faster. However, the cause of this phenomenon remains elusive. Our research rules out alternative splicing as the cause and, instead, identifies the presence of a P124A mutation in SRP14 (SRP14 P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14 P124 or SRP14 WT and SRP14 P124A in various cell lines confirms that the P124A mutation indeed leads to faster migration of SRP14. Further mutagenesis analysis shows that the P117A and A121P mutations within the alanine-rich domain at the C-terminus of SRP14 are responsible for migration alterations on SDS-PAGE, whereas mutations outside this domain, such as P39A, Y27F, and T45A, have no such effect. Furthermore, the ectopic expression of SRP14 WT and SRP14 P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14 P124A represents a natural variant of SRP14 and retains comparable functionality. In conclusion, the substitution of proline for alanine in the alanine-rich tail of SRP14 results in faster migration on SDS-PAGE, but has little effect on its function.

A signal recognition particle receptor gene from the sea cucumber, Apostichopus japonicas.

The signal recognition particle (SRP) system delivers approximately 30% of the proteome to the endoplasmic reticulum (ER) membrane. SRP receptor alpha (SRα) binds to SRP for targeting nascent secreted proteins to the ER membrane in eukaryotic cells. In this study, the SRα homologous gene was identified in the sea cucumber, Apostichopus japonicus (AjSRα). AjSRα codes for 641 amino acids and has 54.94% identity with its mammalian homologs. Like mammalian SRα, it is expected to contain the SRP-alpha N domain, SRP54_N domain, and SRP54 domain. In addition, AjSRα is ubiquitously expressed in adult tissues and exhibits a sexually dimorphic expression pattern, with significantly higher expression in ovaries compared to testes. As a maternal factor, AjSRα can be continuously detected during embryonic development. Importantly, we first attempted to investigate its function by using lentiviral vectors for delivering SRα gene-specific shRNA, and we revealed that lentiviral vectors do not induce an upregulation of immune-related enzymes in sea cucumbers. However, compared to the dsRNA-based RNA interference (RNAi) method, lentivirus-mediated RNAi caused dynamic changes in gene expression at a later time. This study supplied the technical support for studying the functional mechanism of SRα in sea cucumbers.

Examining SRP pathway function in mRNA localization to the endoplasmic reticulum.

Signal recognition particle (SRP) pathway function in protein translocation across the endoplasmic reticulum (ER) is well established; its role in RNA localization to the ER remains, however, unclear. In current models, mRNAs undergo translation- and SRP-dependent trafficking to the ER, with ER localization mediated via interactions between SRP-bound translating ribosomes and the ER-resident SRP receptor (SR), a heterodimeric complex comprising SRA, the SRP-binding subunit, and SRB, an integral membrane ER protein. To study SRP pathway function in RNA localization, SR knockout (KO) mammalian cell lines were generated and the consequences of SR KO on steady-state and dynamic mRNA localization examined. CRISPR/Cas9-mediated SRPRB KO resulted in profound destabilization of SRA. Pairing siRNA silencing of SRPRA in SRPRB KO cells yielded viable SR KO cells. Steady-state mRNA compositions and ER-localization patterns in parental and SR KO cells were determined by cell fractionation and deep sequencing. Notably, steady-state cytosol and ER mRNA compositions and partitioning patterns were largely unaltered by loss of SR expression. To examine SRP pathway function in RNA localization dynamics, the subcellular trafficking itineraries of newly exported mRNAs were determined by 4-thiouridine (4SU) pulse-labeling/4SU-seq/cell fractionation. Newly exported mRNAs were distinguished by high ER enrichment, with ER localization being SR-independent. Intriguingly, under conditions of translation initiation inhibition, the ER was the default localization site for all newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from the SRP pathway function and reopen questions regarding the mechanism of RNA localization to the ER.

Alu RNA fold links splicing with signal recognition particle proteins.

Transcriptomic diversity in primates was considerably expanded by exonizations of intronic Alu elements. To better understand their cellular mechanisms we have used structure-based mutagenesis coupled with functional and proteomic assays to study the impact of successive primate mutations and their combinations on inclusion of a sense-oriented AluJ exon in the human F8 gene. We show that the splicing outcome was better predicted by consecutive RNA conformation changes than by computationally derived splicing regulatory motifs. We also demonstrate an involvement of SRP9/14 (signal recognition particle) heterodimer in splicing regulation of Alu-derived exons. Nucleotide substitutions that accumulated during primate evolution relaxed the conserved left-arm AluJ structure including helix H1 and reduced the capacity of SRP9/14 to stabilize the closed Alu conformation. RNA secondary structure-constrained mutations that promoted open Y-shaped conformations of the Alu made the Alu exon inclusion reliant on DHX9. Finally, we identified additional SRP9/14 sensitive Alu exons and predicted their functional roles in the cell. Together, these results provide unique insights into architectural elements required for sense Alu exonization, identify conserved pre-mRNA structures involved in exon selection and point to a possible chaperone activity of SRP9/14 outside the mammalian signal recognition particle.

Chloroplast SRP43 and SRP54 independently promote thermostability and membrane binding of light-dependent protochlorophyllide oxidoreductases.

Protochlorophyllide oxidoreductase (POR), which converts protochlorophyllide into chlorophyllide, is the only light-dependent enzyme in chlorophyll biosynthesis. While its catalytic reaction and importance for chloroplast development are well understood, little is known about the post-translational control of PORs. Here, we show that cpSRP43 and cpSRP54, two components of the chloroplast signal recognition particle pathway, play distinct roles in optimizing the function of PORB, the predominant POR isoform in Arabidopsis. The chaperone cpSRP43 stabilizes the enzyme and provides appropriate amounts of PORB during leaf greening and heat shock, whereas cpSRP54 enhances its binding to the thylakoid membrane, thereby ensuring adequate levels of metabolic flux in late chlorophyll biosynthesis. Furthermore, cpSRP43 and the DnaJ-like protein CHAPERONE-LIKE PROTEIN of POR1 concurrently act to stabilize PORB. Overall, these findings enhance our understanding of the coordinating role of cpSPR43 and cpSRP54 in the post-translational control of chlorophyll synthesis and assembly of photosynthetic chlorophyll-binding proteins.

Nuclear SRP9/SRP14 heterodimer transcriptionally regulates 7SL and BC200 RNA expression.

The SRP9/SRP14 heterodimer is a central component of signal recognition particle (SRP) RNA (7SL) processing and Alu retrotransposition. In this study, we sought to establish the role of nuclear SRP9/SRP14 in the transcriptional regulation of 7SL and BC200 RNA. 7SL and BC200 RNA steady-state levels, rate of decay, and transcriptional activity were evaluated under SRP9/SRP14 knockdown conditions. Immunofluorescent imaging, and subcellular fractionation of MCF-7 cells, revealed a distinct nuclear localization for SRP9/SRP14. The relationship between this localization and transcriptional activity at 7SL and BC200 genes was also examined. These findings demonstrate a novel nuclear function of SRP9/SRP14 establishing that this heterodimer transcriptionally regulates 7SL and BC200 RNA expression. We describe a model in which SRP9/SRP14 cotranscriptionally regulate 7SL and BC200 RNA expression. Our model is also a plausible pathway for regulating Alu RNA transcription and is consistent with the hypothesized roles of SRP9/SRP14 transporting 7SL RNA into the nucleolus for posttranscriptional processing, and trafficking of Alu RNA for retrotransposition.

Interaction of FlhF, SRP-like GTPase with FliF, MS ring component assembling the initial structure of flagella in marine Vibrio.

Vibrio alginolyticus forms a single flagellum at its cell pole. FlhF and FlhG are known to be the main proteins responsible for the polar formation of single flagellum. MS-ring formation in the flagellar basal body appears to be an initiation step for flagellar assembly. The MS-ring is formed by a single protein, FliF, which has two transmembrane (TM) segments and a large periplasmic region. We had shown that FlhF was required for the polar localization of Vibrio FliF, and FlhF facilitated MS-ring formation when FliF was overexpressed in Escherichia coli cells. These results suggest that FlhF interacts with FliF to facilitate MS-ring formation. Here, we attempted to detect this interaction using Vibrio FliF fragments fused to a tag of Glutathione S-transferase in E. coli. We found that the N-terminal 108 residues of FliF, including the first TM segment and the periplasmic region, could pull FlhF down. In the first step, signal recognition particle (SRP) and its receptor are involved in the transport of membrane proteins to target them, which delivers them to the translocon. FlhF may have a similar or enhanced function as SRP, which binds to a region rich in hydrophobic residues.

Mechanism of signal-anchor triage during early steps of membrane protein insertion.

Most membrane proteins use their first transmembrane domain, known as a signal anchor (SA), for co-translational targeting to the endoplasmic reticulum (ER) via the signal recognition particle (SRP). The SA then inserts into the membrane using either the Sec61 translocation channel or the ER membrane protein complex (EMC) insertase. How EMC and Sec61 collaborate to ensure SA insertion in the correct topology is not understood. Using site-specific crosslinking, we detect a pre-insertion SA intermediate adjacent to EMC. This intermediate forms after SA release from SRP but before ribosome transfer to Sec61. The polypeptide's N-terminal tail samples a cytosolic vestibule bordered by EMC3, from where it can translocate across the membrane concomitant with SA insertion. The ribosome then docks on Sec61, which has an opportunity to insert those SAs skipped by EMC. These results suggest that EMC acts between SRP and Sec61 to triage SAs for insertion during membrane protein biogenesis.

mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria.

Signal-sequence-dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and is facilitated by dedicated protein targeting factors such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors but is dependent on the SecYEG translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised.

Fine mapping of CscpFtsY, a gene conferring the yellow leaf phenotype in cucumber (Cucumis sativus L.).

BACKGROUND: Leaf color mutants are ideal materials to study pigment metabolism and photosynthesis. Leaf color variations are mainly affected by chlorophylls (Chls) and carotenoid contents and chloroplast development in higher plants. However, the regulation of chlorophyll metabolism remains poorly understood in many plant species. The chloroplast signal-recognition particle system is responsible for the insertion of the light-harvesting chlorophyll a/b proteins (LHCPs) to thylakoid membranes, which controls the chloroplast development as well as the regulation of Chls biosynthesis post-translationally in higher plants. RESULTS: In this study, the yellow leaf cucumber mutant, named yl, was found in an EMS-induced mutant library, which exhibited a significantly reduced chlorophyll content, abnormal chloroplast ultrastructure and decreased photosynthetic capacity. Genetic analysis demonstrated that the phenotype of yl was controlled by a recessive nuclear gene. Using BSA-seq technology combined with the map-based cloning method, we narrowed the locus to a 100 kb interval in chromosome 3. Linkage analysis and allelism test validated the candidate SNP residing in CsaV3_3G009150 encoding one homolog of chloroplast signal-recognition particle (cpSRP) receptor in Arabidopsis, cpFtsY, could be responsible for the yellow leaf phenotype of yl. The relative expression of CscpFtsY was significantly down-regulated in different organs except for the stem, of yl compared with that in the wild type (WT). Subcellular localization result showed that CscpFtsY located in the chloroplasts of mesophyll cells. CONCLUSIONS: The yl mutant displayed Chls-deficient, impaired chloroplast ultrastructure with intermittent grana stacks and significantly decreased photosynthetic capacity. The isolation of CscpFtsY in cucumber could accelerate the progress on chloroplast development by cpSRP-dependant LHCP delivery system and regulation of Chls biosynthesis in a post-translational way.

Nascent polypeptide-Associated Complex and Signal Recognition Particle have cardiac-specific roles in heart development and remodeling.

Establishing a catalog of Congenital Heart Disease (CHD) genes and identifying functional networks would improve our understanding of its oligogenic underpinnings. Our studies identified protein biogenesis cofactors Nascent polypeptide-Associated Complex (NAC) and Signal-Recognition-Particle (SRP) as disease candidates and novel regulators of cardiac differentiation and morphogenesis. Knockdown (KD) of the alpha- (Nacα) or beta-subunit (bicaudal, bic) of NAC in the developing Drosophila heart disrupted cardiac developmental remodeling resulting in a fly with no heart. Heart loss was rescued by combined KD of Nacα with the posterior patterning Hox gene Abd-B. Consistent with a central role for this interaction in cardiogenesis, KD of Nacα in cardiac progenitors derived from human iPSCs impaired cardiac differentiation while co-KD with human HOXC12 and HOXD12 rescued this phenotype. Our data suggest that Nacα KD preprograms cardioblasts in the embryo for abortive remodeling later during metamorphosis, as Nacα KD during translation-intensive larval growth or pupal remodeling only causes moderate heart defects. KD of SRP subunits in the developing fly heart produced phenotypes that targeted specific segments and cell types, again suggesting cardiac-specific and spatially regulated activities. Together, we demonstrated directed function for NAC and SRP in heart development, and that regulation of NAC function depends on Hox genes.

Defective Human SRP Induces Protein Quality Control and Triggers Stress Response.

Regulation of Aberrant Protein Production (RAPP) is a protein quality control in mammalian cells. RAPP degrades mRNAs of nascent proteins not able to associate with their natural interacting partners during synthesis at the ribosome. However, little is known about the molecular mechanism of the pathway, its substrates, or its specificity. The Signal Recognition Particle (SRP) is the first interacting partner for secretory proteins. It recognizes signal sequences of the nascent polypeptides when they are exposed from the ribosomal exit tunnel. Here, we reveal the generality of the RAPP pathway on the whole transcriptome level through depletion of human SRP54, an SRP subunit. This depletion triggers RAPP and leads to decreased expression of the mRNAs encoding a number of secretory and membrane proteins. The loss of SRP54 also leads to the dramatic upregulation of a specific network of HSP70/40/90 chaperones (HSPA1A, DNAJB1, HSP90AA1, and others), increased ribosome associated ubiquitination, and change in expression of RPS27 and RPS27L suggesting ribosome rearrangement. These results demonstrate the complex nature of defects in protein trafficking, mRNA and protein quality control, and provide better understanding of their mechanisms at the ribosome.

Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting.

Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals.

Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins.

DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency.

Spatiotemporal kinetics of the SRP pathway in live E. coli cells.

Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of in vitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining in vivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in Escherichia coli, no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons.

Identification and characterization of tsyl1, a thermosensitive chlorophyll-deficient mutant in rice (Oryza sativa).

Chloroplast development and chlorophyll biosynthesis are affected by temperature. However, the underlying molecular mechanism of this phenomenon remains elusive. Here, we isolated and characterized a thermosensitive yellow-green leaf mutant named tsyl1 (thermosensitive yellow leaf 1) from an ethylmethylsulfone (EMS)-mutagenized pool of rice. The mutant exhibits a yellow-green leaf phenotype and decreased leaf chlorophyll contents throughout development. At the mature stage of the tsyl1 mutant, the plant height, tiller number, number of spikelets per panicle and 1000 seed weight were decreased significantly compared to those of wild-type plants, but the seed setting rate and panicle length were not. The mutant phenotype was controlled by a single recessive nuclear gene on the short arm of rice chromosome 11. Map-based cloning of TSYL1, followed by a complementation experiment, showed a G base deletion at the coding region of LOC_Os11g05552, leading to the yellow-green phenotype. The TSYL1 gene encodes a signal recognition particle 54 kDa (SRP54) protein that is conserved in all organisms. The expression of tsyl1 was induced by high temperature. Furthermore, the expression of chlorophyll biosynthesis- and chloroplast development-related genes was influenced in tsyl1 at different temperatures. These results indicated that the TSYL1 gene plays a key role in chlorophyll biosynthesis and is affected by temperature at the transcriptional level.

A 'NAC' for targeting proteins to the ER.

The signal recognition particle (SRP) cotranslationally targets a large and diverse portion of the nascent proteome to the endoplasmic reticulum (ER). A recent study by Jomaa et al. reveals an unexpected function for the ribosome-bound nascent chain-associated complex (NAC) in sensing ER-targeting signals and recruiting SRP to the appropriate ribosomes for high-fidelity targeting.

Expression and purification of the NG domain from human SRα, a key component of the Signal Recognition Particle (SRP) receptor.

The Signal Recognition Particle (SRP) and the SRP receptor (SR) are responsible for protein targeting to the plasma membrane and the protein secretory pathway. Eukaryotic SRα, one of the two proteins that form the SR, is composed of the NG, MoRF and X domains. The SRα-NG domain is responsible for binding to SRP proteins such as SRP54, interacting with RNA, binding and hydrolysing GTP. The ability to produce folded SRα-NG is a prerequisite for structural studies directed towards a better understanding of its molecular mechanism and function, as well as in (counter-)screening assays for potential binders in the drug development pipeline. However, previously reported SRα-NG constructs and purification methods only used a truncated version, lacking the first N-terminal helix. This helix in other NG domains (e.g., SRP54) has been shown to be important for protein:protein interactions but its importance in SRα remains unknown. Here, we present the cloning as well as optimised expression and purification protocols of the whole SRα-NG domain including the first N-terminal helix. We have also expressed and purified isotopically labelled SRα-NG to facilitate Nuclear Magnetic Resonance (NMR) studies.