Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality.

Signal recognition particles (SRPs) are essential for regulating intracellular protein transport and secretion. Patients with tumors with high SRP9 expression tend to have a poorer overall survival. However, to the best of our knowledge, no reports have described the relationship between SRP9 localization and prognosis in pancreatic cancer. Thus, the present study aimed to investigate this relationship. Immunohistochemical staining for SRP9 using excised specimens from pancreatic cancer surgery cases without preoperative chemotherapy or radiotherapy showed that SRP9 was preferentially expressed in the nucleus of the cancerous regions in some cases, which was hardly detected in other cases, indicating that SRP9 was transported to the nucleus in the former cases. To compare the prognosis of patients with SRP9 nuclear translocation, patients were divided into two groups: Those with a nuclear translocation rate of >50% and those with a nuclear translocation rate of ≤50%. The nuclear translocation rate of >50% group had a significantly better recurrence­free survival than the nuclear translocation rate of ≤50% group (P=0.037). Subsequent in vitro experiments were conducted; notably, the nuclear translocation rate of SRP9 was reduced under amino acid­deficient conditions, suggesting that multiple factors are involved in this phenomenon. To further study the function of SRP9 nuclear translocation, in vitro experiments were performed by introducing SRP9 splicing variants (v1 and v2) and their deletion mutants lacking C­terminal regions into MiaPaCa pancreatic cancer cells. The results demonstrated that both splicing variants showed nuclear translocation regardless of the C­terminal deletions, suggesting the role of the N­terminal regions. Given that SRP9 is an RNA­binding protein, the study of RNA immunoprecipitation revealed that signaling pathways involved in cancer progression and protein translation were downregulated in nuclear­translocated v1 and v2. Undoubtedly, further studies of the nuclear translocation of SRP9 will open an avenue to optimize the precise evaluation and therapeutic control of pancreatic cancer.

A Guide to Computational Cotranscriptional Folding Featuring the SRP RNA.

Although RNA molecules are synthesized via transcription, little is known about the general impact of cotranscriptional folding in vivo. We present different computational approaches for the simulation of changing structure ensembles during transcription, including interpretations with respect to experimental data from literature. Specifically, we analyze different mutations of the E. coli SRP RNA, which has been studied comparatively well in previous literature, yet the details of which specific metastable structures form as well as when they form are still under debate. Here, we combine thermodynamic and kinetic, deterministic, and stochastic models with automated and visual inspection of those systems to derive the most likely scenario of which substructures form at which point during transcription. The simulations do not only provide explanations for present experimental observations but also suggest previously unnoticed conformations that may be verified through future experimental studies.

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.

Nonsense-mediated mRNA decay of mRNAs encoding a signal peptide occurs primarily after mRNA targeting to the endoplasmic reticulum.

Translation of messenger ribonucleic acids (mRNAs) encoding integral membrane proteins or secreted proteins occurs on the surface of the endoplasmic reticulum (ER). When a nascent signal peptide is synthesized from the mRNAs, the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP) and then transported to the surface of the ER. The appropriate targeting of the RNC-SRP complex to the ER is monitored by a quality control pathway, a nuclear cap-binding complex (CBC)-ensured translational repression of RNC-SRP (CENTRE). In this study, using ribosome profiling of CBC-associated and eukaryotic translation initiation factor 4E-associated mRNAs, we reveal that, at the transcriptomic level, CENTRE is in charge of the translational repression of the CBC-RNC-SRP until the complex is specifically transported to the ER. We also find that CENTRE inhibits the nonsense-mediated mRNA decay (NMD) of mRNAs within the CBC-RNC-SRP. The NMD occurs only after the CBC-RNC-SRP is targeted to the ER and after eukaryotic translation initiation factor 4E replaces CBC. Our data indicate dual surveillance for properly targeting mRNAs encoding integral membrane or secretory proteins to the ER. CENTRE blocks gene expression at the translation level before the CBC-RNC-SRP delivery to the ER, and NMD monitors mRNA quality after its delivery to the ER.

Whole-Exome Sequencing (WES) Reveals Novel Sex-Specific Gene Variants in Non-Alcoholic Steatohepatitis (MASH).

Non-alcoholic steatohepatitis (NASH, also known as MASH) is a severe form of non-alcoholic fatty liver disease (NAFLD, also known as MASLD). Emerging data indicate that the progression of the disease to MASH is higher in postmenopausal women and that genetic susceptibility increases the risk of MASH-related cirrhosis. This study aimed to investigate the association between genetic polymorphisms in MASH and sexual dimorphism. We applied whole-exome sequencing (WES) to identify gene variants in 8 age-adjusted matched pairs of livers from both male and female patients. Sequencing alignment, variant calling, and annotation were performed using standard methods. Polymerase chain reaction (PCR) coupled with Sanger sequencing and immunoblot analysis were used to validate specific gene variants. cBioPortal and Gene Set Enrichment Analysis (GSEA) were used for actionable target analysis. We identified 148,881 gene variants, representing 57,121 and 50,150 variants in the female and male cohorts, respectively, of which 251 were highly significant and MASH sex-specific (p < 0.0286). Polymorphisms in CAPN14, SLC37A3, BAZ1A, SRP54, MYH11, ABCC1, and RNFT1 were highly expressed in male liver samples. In female samples, Polymorphisms in RGSL1, SLC17A2, HFE, NLRC5, ACTN4, SBF1, and ALPK2 were identified. A heterozygous variant 1151G>T located on 18q21.32 for ALPK2 (rs3809983) was validated by Sanger sequencing and expressed only in female samples. Immunoblot analysis confirmed that the protein level of ß-catenin in female samples was 2-fold higher than normal, whereas ALPK2 expression was 0.5-fold lower than normal. No changes in the protein levels of either ALPK2 or ß-catenin were observed in male samples. Our study suggests that the perturbation of canonical Wnt/ß-catenin signaling observed in postmenopausal women with MASH could be the result of polymorphisms in ALPK2.

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.

Clinical features, epidemiology, and treatment of Shwachman-Diamond syndrome: a systematic review.

BACKGROUND: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease which results in inherited bone marrow failure (IBMF) and is characterized by exocrine pancreatic dysfunction and diverse clinical phenotypes. In the present study, we reviewed the internationally published reports on SDS patients, in order to summarize the clinical features, epidemiology, and treatment of SDS. METHODS: We searched the WangFang and China National Knowledge Infrastructure databases with the keywords "Shwachman-Diamond syndrome," "SDS," "SBDS gene" and "inherited bone marrow failure" for relevant articles published from January 2002 to October 2022. In addition, studies published from January 2002 to October 2022 were searched from the Web of Science, PubMed, and MEDLINE databases, using "Shwachman-diamond syndrome" as the keyword. Finally, one child with SDS treated in Tongji Hospital was also included. RESULTS: The clinical features of 156 patients with SDS were summarized. The three major clinical features of SDS were found to be peripheral blood cytopenia (96.8%), exocrine pancreatic dysfunction (83.3%), and failure to thrive (83.3%). The detection rate of SDS mutations was 94.6% (125/132). Mutations in SBDS, DNAJC21, SRP54, ELF6, and ELF1 have been reported. The male-to-female ratio was approximately 1.3/1. The median age of onset was 0.16 years, but the diagnostic age lagged by a median age of 1.3 years. CONCLUSIONS: Pancreatic exocrine insufficiency and growth failure were common initial symptoms. SDS onset occurred early in childhood, and individual differences were obvious. Comprehensive collection and analysis of case-related data can help clinicians understand the clinical characteristics of SDS, which may improve early diagnosis and promote effective clinical intervention.

Detection of a 7SL RNA-derived small non-coding RNA using Molecular Beacons in vitro and in cells.

Small non-coding RNAs (sncRNA) are involved in many steps of the gene expression cascade and regulate processing and expression of mRNAs by the formation of ribonucleoprotein complexes (RNP) such as the RNA-induced silencing complex (RISC). By analyzing small RNA Seq data sets, we identified a sncRNA annotated as piR-hsa-1254, which is likely derived from the 3'-end of 7SL RNA2 (RN7SL2), herein referred to as snc7SL RNA. The 7SL RNA is an abundant long non-coding RNA polymerase III transcript and serves as structural component of the cytoplasmic signal recognition particle (SRP). To evaluate a potential functional role of snc7SL RNA, we aimed to define its cellular localization by live cell imaging. Therefore, a Molecular Beacon (MB)-based method was established to compare the subcellular localization of snc7SL RNA with its precursor 7SL RNA. We designed and characterized several MBs in vitro and tested those by live cell fluorescence microscopy. Using a multiplex approach, we show that 7SL RNA localizes mainly to the endoplasmic reticulum (ER), as expected for the SRP, whereas snc7SL RNA predominately localizes to the nucleus. This finding suggests a fundamentally different function of 7SL RNA and its derivate snc7SL RNA.

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.

Shwachman-Diamond syndromes: clinical, genetic, and biochemical insights from the rare variants.

Shwachman-Diamond syndrome is a rare inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. In 10-30% of cases, transformation to a myeloid neoplasm occurs. Approximately 90% of patients have biallelic pathogenic variants in the SBDS gene located on human chromosome 7q11. Over the past several years, pathogenic variants in three other genes have been identified to cause similar phenotypes; these are DNAJC21, EFL1, and SRP54. Clinical manifestations involve multiple organ systems and those classically associated with the Shwachman-Diamond syndrome (bone, blood, and pancreas). Neurocognitive, dermatologic, and retinal changes may also be found. There are specific gene-phenotype differences. To date, SBDS, DNAJC21, and SRP54 variants have been associated with myeloid neoplasia. Common to SBDS, EFL1, DNAJC21, and SRP54 is their involvement in ribosome biogenesis or early protein synthesis. These four genes constitute a common biochemical pathway conserved from yeast to humans that involve early stages of protein synthesis and demonstrate the importance of this synthetic pathway in myelopoiesis.

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.

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.

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.

Severe congenital neutropenia, SRP54 pathogenicity, and a framework for surveillance.

Severe congenital neutropenia (SCN) is a rare disorder, often due to pathogenic variants in genes such as ELANE, HAX1, and SBDS. SRP54 pathogenic variants are associated with SCN and Shwachman-Diamond-like syndrome. Thirty-eight patients with SRP54-related SCN are reported in the literature. We present an infant with SCN, without classic Shwachman-Diamond syndrome features, who presented with recurrent bacterial infections and an SRP54 (c.349_351del) pathogenic variant. Despite ongoing granulocyte colony-stimulating factor therapy, this patient has no evidence of malignant transformation. Here we establish a framework for the future development of universal guidelines to care for this patient population.

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.

Case Report: Association between cyclic neutropenia and SRP54 deficiency.

Autosomal dominant mutations in the signal recognition particle (SRP) 54 gene were recently described in patients with severe congenital neutropenia (SCN). SRP54 deficiency cause a chronic and profound neutropenia with maturation arrest at the promyelocyte stage, occurring in the first months of life. Nearly all reported patients with SRP54 mutations had neutropenia without a cyclic pattern and showed a poor or no response to granulocyte colony-stimulating factor (G-CSF) therapy. We report here an 11-year-old female patient with cyclic neutropenia and recurrent heterozygous p.T117del (c.349_351del) in-frame deletion mutation in SRP54, who showed remarkable therapeutic response to G-CSF treatment. The diagnosis of cyclic pattern of neutropenia was established by acceptable standards. ELANE gene mutation was excluded by using various genetic approaches. The patient described here also had dolichocolon which has not been described before in association with SCN.

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.

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.