LARP1, an RNA-binding protein, participates in ovarian cancer cell survival by regulating mitochondrial oxidative phosphorylation in response to the influence of the PI3K/mTOR pathway.

Targeting the PI3K/mTOR pathway and modulating mitochondrial adaptation is expected to be a critical approach for cancer therapy. Although the regulation of mitochondria by the PI3K/mTOR pathway has been investigated, it is not well understood due to the complexity of its regulatory mechanisms. RNA-binding proteins (RBPs) selectively regulate gene expression through post-transcriptional modulation, playing a key role in cancer progression. LARP1, a downstream RBP of the mTOR pathway, is involved in mitochondria-mediated BCL-2 cell survival. Therefore, exploring the involvement of LARP1 in PI3K/mTOR-mediated translational regulation of mitochondria-associated proteins in ovarian cancer cells could help elucidate the role of mitochondria in the PI3K/mTOR pathway. We found that, unlike SKOV3 cells, the mitochondrial function of A2780 cells was not affected, which were insensitive to the dual PI3K/mTOR inhibitor PKI-402, suggesting that cell survival may be related to mitochondrial function. Knockdown of the LARP1 gene after PKI-402 treatment resulted in impaired mitochondrial function in A2780 cells, possibly due to decreased mRNA stability and reduced protein translation of the mitochondrial transcription initiation factor, TFB2M, and the respiratory chain complex II subunit, SDHB. LARP1 affects protein translation by binding to TFB2M mRNA, regulating mitochondrial DNA-encoded genes, or indirectly regulating the nuclear DNA-encoded SDHB gene, ultimately interfering with mitochondrial oxidative phosphorylation and leading to apoptosis. Therefore, LARP1 may be an important mediator in the PI3K/mTOR pathway for regulating mRNA translation and mitochondrial function. Targeting RBPs such as LARP1 downstream of the mTOR pathway may provide new insights and potential therapeutic approaches for ovarian cancer treatment.

Comparative analysis of the LARP1 C-terminal DM15 region through Coelomate evolution.

TOR (target of rapamycin), a ubiquitous protein kinase central to cellular homeostasis maintenance, fundamentally regulates ribosome biogenesis in part by its target La-related protein 1 (LARP1). Among other target transcripts, LARP1 specifically binds TOP (terminal oligopyrimidine) mRNAs encoding all 80 ribosomal proteins in a TOR-dependent manner through its C-terminal region containing the DM15 module. Though the functional implications of the LARP1 interaction with target mRNAs is controversial, it is clear that the TOP-LARP1-TOR axis is critical to cellular health in humans. Its existence and role in evolutionarily divergent animals remain less understood. We focused our work on expanding our knowledge of the first arm of the axis: the connection between LARP1-DM15 and the 5' TOP motif. We show that the overall DM15 architecture observed in humans is conserved in fruit fly and zebrafish. Both adopt familiar curved arrangements of HEAT-like repeats that bind 5' TOP mRNAs on the same conserved surface, although molecular dynamics simulations suggest that the N-terminal fold of the fruit fly DM15 is predicted to be unstable and unfold. We demonstrate that each ortholog interacts with TOP sequences with varying affinities. Importantly, we determine that the ability of the DM15 region to bind some TOP sequences but not others might amount to the context of the RNA structure, rather than the ability of the module to recognize some sequences but not others. We propose that TOP mRNAs may retain similar secondary structures to regulate LARP1 DM15 recognition.

Adolescent co-exposure to environmental cadmium and high-fat diet induces cognitive decline via Larp7 m6A-mediated SIRT6 inhibition.

The effects and underlying mechanisms of adolescent exposure to combined environmental hazards on cognitive function remain unclear. Here, using a combined exposure model, we found significant cognitive decline, hippocampal neuronal damage, and neuronal senescence in mice exposed to cadmium (Cd) and high-fat diet (HFD) during adolescence. Furthermore, we observed a significant downregulation of Sirtuin 6 (SIRT6) expression in the hippocampi of co-exposed mice. UBCS039, a specific SIRT6 activator, markedly reversed the above adverse effects. Further investigation revealed that co-exposure obviously reduced the levels of La ribonucleoprotein 7 (LARP7), disrupted the interaction between LARP7 and SIRT6, ultimately decreasing SIRT6 expression in mouse hippocampal neuronal cells. Overexpression of Larp7 reversed the combined exposure-induced SIRT6 decrease and senescence in mouse hippocampal neuronal cells. Additionally, the results showed notably elevated levels of Larp7 m6A and YTH domain family protein 2 (YTHDF2) in mouse hippocampal neuronal cells treated with the combined hazards. Ythdf2 short interfering RNA, RNA immunoprecipitation, and RNA stability assays further demonstrated that YTHDF2 mediated the degradation of Larp7 mRNA under combined exposure. Collectively, adolescent co-exposure to Cd and HFD causes hippocampal senescence and cognitive decline in mice by inhibiting LARP7-mediated SIRT6 expression in an m6A-dependent manner.

Enhanced binding of guanylated poly(A) RNA by the LaM domain of LARP1.

La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].

LARP7 Contributes to Glucose-Induced Cardiac Dysfunction, Apoptosis and Fibrosis by Inhibiting the Degradation of STING.

BACKGROUND: Diabetic cardiomyopathy (DCM) is an important cause of heart failure in diabetic patients. The aim of this study was to investigate the pathogenesis of DCM and to identify potential therapeutic targets. METHODS: A mouse model of type 1 DCM was constructed by continuous intraperitoneal injection of streptozotocin (STZ). Systolic and diastolic functions were measured by ultrasound. The expression of La-related protein 7 (LARP7), the stimulator of interferon genes (STING) pathway and light chain 3 (LC3) in myocardial tissue was detected by Western blot and immunofluorescence analyses. Neonatal mouse ventricular cardiomyocytes (NMVCMs) were isolated and cultured. An in vitro type 1 diabetes mellitus (T1DM) model was established by treatment with high glucose. Knockdown/overexpression of LARP7 and STING was achieved by adenovirus transduction, C-176 (a potent covalent inhibitor of STING), and plasmid transfection. The expression, activation, and localization of STING and LARP7 in cardiomyocytes was evaluated, as well as the interaction between the two. The effect of this interaction on the STING-dependent autophagy‒lysosomal pathway was also explored. In addition, the fibrosis and apoptosis of cardiomyocytes were evaluated. RESULTS: High glucose was found to increase the expression and activation of STING and LARP7 in mouse myocardial tissue. This was accompanied by myocardial fibrosis, impaired autophagy degradation function and impaired cardiac function. These findings were further confirmed by in vitro experiments. High glucose caused LARP7 to translocate from the nucleus to the cytoplasm, where it interacted with accumulated STING to inhibit its degradation. Inhibition of STING or LARP7 expression significantly improved myocardial injury induced by high glucose. CONCLUSIONS: Targeted inhibition of LARP7 or STING expression may be a potential therapeutic strategy for the treatment of DCM.

LARP3, LARP7, and MePCE are involved in the early stage of human telomerase RNA biogenesis.

Human telomerase assembly is a highly dynamic process. Using biochemical approaches, we find that LARP3 and LARP7/MePCE are involved in the early stage of human telomerase RNA (hTR) and that their binding to RNA is destabilized when the mature form is produced. LARP3 plays a negative role in preventing the processing of the 3'-extended long (exL) form and the binding of LARP7 and MePCE. Interestingly, the tertiary structure of the exL form prevents LARP3 binding and facilitates hTR biogenesis. Furthermore, low levels of LARP3 promote hTR maturation, increase telomerase activity, and elongate telomeres. LARP7 and MePCE depletion inhibits the conversion of the 3'-extended short (exS) form into mature hTR and the cytoplasmic accumulation of hTR, resulting in telomere shortening. Taken together our data suggest that LARP3 and LARP7/MePCE mediate the processing of hTR precursors and regulate the production of functional telomerase.

A novel protein encoded by circLARP1B promotes the proliferation and migration of vascular smooth muscle cells by suppressing cAMP signaling.

BACKGROUND AND AIMS: Circular RNA (circRNA) is closely related to atherosclerosis (AS) incidence and progression, but its regulatory mechanism in AS needs further elucidation. AS development is significantly influenced by abnormal vascular smooth muscle cells (VSMCs) growth and migration. This study explored the potential protein role of circLARP1B in VSMC proliferation and migration. METHODS: We performed whole-transcriptome sequencing in human normal arterial intima and advanced atherosclerotic plaques to screen for differentially expressed circRNAs. The sequencing results were combined with database analysis to screen for circRNAs with coding ability. Real-time quantitative polymerase chain reaction was utilized to assess circLARP1B expression levels in atherosclerotic plaque tissues and cells. circLARP1B-243aa function and pathway in VSMCs growth and migration were studied by scratch, transwell, 5-ethynyl-2'-deoxyuridine, cell counting kit-8, and Western blot experiments. RESULTS: We found that circLARP1B was downregulated in atherosclerotic plaque tissue and promoted the proliferation and migration of VSMCs. circLARP1B encodes a novel protein with a length of 243 amino acids. Through functional experiments, we confirmed the role of circLARP1B-243aa in enhancing VSMCs migration and proliferation. Mechanistically, circLARP1B-243aa promotes VSMCs migration and growth by upregulating phosphodiesterase 4C to inhibit the cyclic adenosine monophosphate signaling pathway. CONCLUSIONS: Our results suggested that circLARP1B could promote VSMCs growth and migration through the encoded protein circLARP1B-243aa. Therefore, it could be a treatment target and biomarker for AS.

The binding of LARP6 and DNAAF6 in biomolecular condensates influences ciliogenesis of multiciliated cells.

Motile cilia on the cell surface produce fluid flows in the body and abnormalities in motile cilia cause primary ciliary dyskinesia. Dynein axonemal assembly factor 6 (DNAAF6), a causative gene of primary ciliary dyskinesia, was isolated as an interacting protein with La ribonucleoprotein 6 (LARP6) that regulates ciliogenesis in multiciliated cells (MCCs). In MCCs of Xenopus embryos, LARP6 and DNAAF6 were colocalized in biomolecular condensates termed dynein axonemal particles and synergized to control ciliogenesis. Moreover, tubulin alpha 1c-like mRNA encoding α-tubulin protein, that is a major component of ciliary axoneme, was identified as a target mRNA regulated by binding LARP6. While DNAAF6 was necessary for high α-tubulin protein expression near the apical side of Xenopus MCCs during ciliogenesis, its mutant, which abolishes binding with LARP6, was unable to restore the expression of α-tubulin protein near the apical side of MCCs in Xenopus DNAAF6 morphant. These results indicated that the binding of LARP6 and DNAAF6 in dynein axonemal particles regulates highly expressed α-tubulin protein near the apical side of Xenopus MCCs during ciliogenesis.

Biological functions of Circular RNA_LARP4/ Upstream frameshift 1 in development of gastric cancer.

Recently, the progression of gastric cancer (GC), as one of the most ordinary malignant tumors, has been reported to be associated with circular RNAs. This study aimed to identify the role of circular RNA_LARP4 in GC. We performed real-time quantitative polymerase chain reaction (RT-qPCR) in 46 paired GC patients and GC cell lines to detect the expression of circular RNA_LARP4. Moreover, the role of circular RNA_LARP4 in GC proliferation was identified through proliferation assay and colony formation assay, while the role of circular RNA_LARP4 in GC metastasis was measured through scratch wound assay and transwell assay. Furthermore, the potential targets of circular RNA_LARP4 were predicted through bioinformatics methods and further identified by western blot assay and RT-qPCR. Circular RNA_LARP4 expression was remarkably lower in GC tissues compared with that in adjacent samples. Besides, cell proliferation of GC was inhibited after overexpression of circular RNA_LARP4, while cell migration and invasion of GC was inhibited after overexpression of circular RNA_LARP4. Furthermore, Upstream frameshift 1 (UPF1) was predicted as the potential target of circular RNA_LARP4 and was upregulated via overexpression of circular RNA_LARP4 in GC. Circular RNA_LARP4 inhibits GC cell proliferation and metastasis via targeting UPF1 in vitro, which might provide a new tumor suppressor in GC development.

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry.

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

O-GlcNAcylated LARP1 positively regulated by circCLNS1A facilitates hepatoblastoma progression through DKK4/ß-catenin signalling.

BACKGROUND: Accumulating studies have shown that La-related protein 1 (LARP1) is involved in the occurrence and development of various tumours. However, the expression pattern and biological role of LARP1 in hepatoblastoma (HB) remain unclear so far. METHODS: LARP1 expression level in HB and adjacent normal liver tissues was analysed by qRT-PCR, Western blotting and immunohistochemistry assays. The prognostic significance of LARP1 was evaluated by Kaplan-Meier method and multivariate Cox regression analysis. In vitro and in vivo functional assays were implemented to clarify the biological effects of LARP1 on HB cells. Mechanistically, the regulatory roles of O-GlcNAcylation and circCLNS1A in LARP1 expression were investigated by co-immunoprecipitation (co-IP), immunofluorescence, RNA immunoprecipitation (RIP), RNA pull-down and protein stability assays. Moreover, RNA-sequencing, co-IP, RIP, mRNA stability and poly(A)-tail length assays were performed to investigate the association between LARP1 and DKK4. The expression and diagnostic significance of plasma DKK4 protein in multi-centre cohorts were evaluated by ELISA and ROC curves. RESULTS: LARP1 mRNA and protein levels were remarkably elevated in HB tissues and associated with worse prognosis of HB patients. LARP1 knockdown abolished cell proliferation, triggered cell apoptosis in vitro as well as prohibited tumour growth in vivo, whereas LARP1 overexpression incited HB progression. Mechanistically, O-GlcNAcylation of LARP1 Ser672 by O-GlcNAc transferase strengthened its binding to circCLNS1A and then protected LARP1 from TRIM-25-mediated ubiquitination and proteolysis. LARP1 upregulation subsequently led to DKK4 mRNA stabilisation by competitively interacting with PABPC1 to prevent DKK4 mRNA from B-cell translocation gene 2-dependent deadenylation and degradation, thus facilitating ß-catenin protein expression and nuclear import. CONCLUSION: This study indicates that upregulated protein level of O-GlcNAcylated LARP1 mediated by circCLNS1A promotes the tumorigenesis and progression of HB through LARP1/DKK4/ß-catenin axis. Hence, LARP1 and DKK4 are promising therapeutical target and diagnostic/prognostic plasma biomarker for HB.

CircBNC2 affects epithelial ovarian cancer progression through the miR-223-3p/ LARP4 axis.

Epithelial ovarian cancer (EOC) is one of the most serious cancer. Circular RNA BNC2 (circBNC2) expression was decreased in EOC tissues. However, the molecular mechanism of circBNC2 remains unknown. The expression of circBNC2, microRNA-223-3p (miR-223-3p), and La-related proteins 4 ( LARP4 ) were detected by quantitative real-time fluorescence PCR (qRT-PCR). A series of in-vitro experiments were designed to explore the function of circBNC2 in EOC cells and the regulatory mechanism between circBNC2 and miR-223-3p and LARP4 in EOC cells. Western blot examined the protein levels of Snail1, Slug, and LARP4 . The relationship between miR-223-3p and circBNC2 or LARP4 was verified by Dual-luciferase reporter assays. The xenotransplantation model was established to study the role of circBNC2 in vivo . The expression of circBNC2 and LARP4 was decreased in EOC tissues, while the expression of miR-223-3p was increased. CircBNC2 can sponge miR-223-3p, and LARP4 is the target of miR-223-3p. In-vitro complement experiments showed that overexpression of circBNC2 significantly decreased the malignant behavior of EOC, while co-transfection of miR-223-3p mimics partially upregulated this change. In addition, LARP4 knockdown increased the proliferation, migration, and invasion of EOC cells inhibited by miR-223-3p inhibitor. Mechanically, circBNC2 regulates LARP4 expression in EOC cells by spongy miR-223-3p. In addition, in-vivo studies have shown that overexpression of circBNC2 inhibits tumor growth. Overexpression of circBNC2 decreased proliferation, migration, and invasion of EOC cells by regulating the miR-223-3p/ LARP4 axis, suggesting that circBNC2/miR-223-3p/ LARP4 axis may be a potential regulatory mechanism for the treatment of EOC.

LARP6 suppresses colorectal cancer progression through ZNF267/SGMS2-mediated imbalance of sphingomyelin synthesis.

BACKGROUND: With increasing incidence and mortality, colorectal cancer (CRC) seriously endangers human health. LARP6, a member of La-related protein (LARP) family, is a RNA binding protein and probably associates with CRC progression, but its specific roles and mechanisms in CRC still remain unknown. METHOD: Quantitative real-time PCR (qPCR), western blot, and immunohistochemistry were employed to examine LARP6 expression in CRC tissues. Using the stable LARP6 overexpression or interference CRC cell lines, the effect of LARP6 on CRC progression were evaluated. High-throughput RNA immunoprecipitation sequencing (RIP-seq) and a series of relevant experiments were conducted to explain how LARP6 functions. SPSS software was used for statistical analysis. RESULT: In this study, we found that LARP6 expression is downregulated in CRC and correlates with patients' overall survival and relapse-free survival. Furthermore, altered LARP6 expression influences CRC cells invasion and metastasis. Mechanically, we discovered that LARP6 bind ZNF267 mRNA and regulated its stability and translation. LARP6 inhibited expression of SGMS2, a downstream target of ZNF267, resulting in ceramide and sphingomyelin imbalance in CRC cells. Interestingly, LARP6 also enhances autophagy activity of CRC cells, and the effect was at least partially determined by the inhibition of SGMS2-mediated sphingomyelin synthesis. CONCLUSION: Our study showed how LARP6/ZNF267/SGMS2 axis influence CRC progression, which contributes to further understanding of the molecular mechanisms underlying CRC development.

Global analysis of RNA-binding proteins identifies a positive feedback loop between LARP1 and MYC that promotes tumorigenesis.

In addition to genomic alterations, aberrant changes in post-transcriptional regulation can modify gene function and drive cancer development. RNA-binding proteins (RBPs) are a large class of post-transcriptional regulators that have been increasingly implicated in carcinogenesis. By integrating multi-omics data, we identify LARP1 as one of the most upregulated RBPs in colorectal cancer (CRC) and demonstrate its oncogenic properties. We perform LARP1:RNA interactome profiling and unveil a previously unexplored role for LARP1 in targeting the 3'UTR of oncogenes in CRC. Notably, we identify the proto-oncogenic transcription factor MYC as a key LARP1-regulated target. Our data show that LARP1 positively modulates MYC expression by associating with its 3'UTR. In addition, antisense oligonucleotide-mediated blocking of the interaction between LARP1 and the MYC 3'UTR reduces MYC expression and in vitro CRC growth. Furthermore, a systematic analysis of LARP1:protein interactions reveals IGF2BP3 and YBX1 as LARP1-interacting proteins that also regulate MYC expression and CRC development. Finally, we demonstrate that MYC reciprocally modulates LARP1 expression by targeting its enhancer. In summary, our data reveal a critical, previously uncharacterized role of LARP1 in promoting CRC tumorigenesis, validate its direct regulation of the proto-oncogene MYC and delineate a model of the positive feedback loop between MYC and LARP1 that promotes CRC growth and development.

Genetic associations with carotid intima-media thickness link to atherosclerosis with sex-specific effects in sub-Saharan Africans.

Atherosclerosis precedes the onset of clinical manifestations of cardiovascular diseases (CVDs). We used carotid intima-media thickness (cIMT) to investigate genetic susceptibility to atherosclerosis in 7894 unrelated adults (3963 women, 3931 men; 40 to 60 years) resident in four sub-Saharan African countries. cIMT was measured by ultrasound and genotyping was performed on the H3Africa SNP Array. Two new African-specific genome-wide significant loci for mean-max cIMT, SIRPA (p = 4.7E-08), and FBXL17 (p = 2.5E-08), were identified. Sex-stratified analysis revealed associations with one male-specific locus, SNX29 (p = 6.3E-09), and two female-specific loci, LARP6 (p = 2.4E-09) and PROK1 (p = 1.0E-08). We replicate previous cIMT associations with different lead SNPs in linkage disequilibrium with SNPs primarily identified in European populations. Our study find significant enrichment for genes involved in oestrogen response from female-specific signals. The genes identified show biological relevance to atherosclerosis and/or CVDs, sex-differences and transferability of signals from non-African studies.

LARP6 Regulates Keloid Fibroblast Proliferation, Invasion, and Ability to Synthesize Collagen.

Keloid is a skin fibroproliferative disease currently having no uniformly successful treatment. The lesion is composed of actively proliferating and collagen-overproducing fibroblasts. LARP6 is an RNA-binding protein able to regulate collagen synthesis in fibroblasts and to promote proliferation and invasion of tumor cells. To explore LARP6's likely functions in keloid pathogenesis, we performed immunohistochemistry staining on human keloid tissues and discovered markedly upregulated LARP6 expression in lesion fibroblasts compared with that of normal skin and hypertrophic scar tissues. In addition, the keloid tissue‒derived fibroblasts showed constitutive upregulation of LARP6 expression as well as significantly upregulated mRNA and protein expressions of type I collagen and enhanced cell proliferation and invasive behavior in cell culture system. Intriguingly, LARP6 knockdown by targeting with small interfering RNAs significantly inhibited type I collagen expression, proliferation, and invasion capability of keloid tissue‒derived fibroblasts relative to that of normal skin‒ and hypertrophic scar‒derived fibroblasts and control keloid tissue‒derived fibroblasts that were transfected with a scrambled small interfering RNA. In conclusion, the abnormally upregulated expression of LARP6 in fibroblasts may play an important role in the growth and invasive behavior of keloid lesions.

S-adenosylmethionine inhibits la ribonucleoprotein domain family member 1 in murine liver and human liver cancer cells.

BACKGROUND AND AIMS: Methionine adenosyltransferase 1A (MAT1A) is responsible for S-adenosylmethionine (SAMe) biosynthesis in the liver. Mice lacking Mat1a have hepatic SAMe depletion and develop NASH and HCC spontaneously. Several kinases are activated in Mat1a knockout (KO) mice livers. However, characterizing the phospho-proteome and determining whether they contribute to liver pathology remain open for study. Our study aimed to provide this knowledge. APPROACH AND RESULTS: We performed phospho-proteomics in Mat1a KO mice livers with and without SAMe treatment to identify SAMe-dependent changes that may contribute to liver pathology. Our studies used Mat1a KO mice at different ages treated with and without SAMe, cell lines, in vitro translation and kinase assays, and human liver specimens. We found that the most striking change was hyperphosphorylation and increased content of La-related protein 1 (LARP1), which, in the unphosphorylated form, negatively regulates translation of 5'-terminal oligopyrimidine (TOP)-containing mRNAs. Consistently, multiple TOP proteins are induced in KO livers. Translation of TOP mRNAs ribosomal protein S3 and ribosomal protein L18 was enhanced by LARP1 overexpression in liver cancer cells. We identified LARP1-T449 as a SAMe-sensitive phospho-site of cyclin-dependent kinase 2 (CDK2). Knocking down CDK2 lowered LARP1 phosphorylation and prevented LARP1-overexpression-mediated increase in translation. LARP1-T449 phosphorylation induced global translation, cell growth, migration, invasion, and expression of oncogenic TOP-ribosomal proteins in HCC cells. LARP1 expression is increased in human NASH and HCC. CONCLUSIONS: Our results reveal a SAMe-sensitive mechanism of LARP1 phosphorylation that may be involved in the progression of NASH to HCC.

The RNA-binding protein La/SSB associates with radiation-induced DNA double-strand breaks in lung cancer cell lines.

BACKGROUND: Platinum-based chemotherapy and radiotherapy are standard treatments for non-small cell lung cancer, which is the commonest, most lethal cancer worldwide. As a marker of treatment-induced cancer cell death, we have developed a radiodiagnostic imaging antibody, which binds to La/SSB. La/SSB is an essential, ubiquitous ribonuclear protein, which is over expressed in cancer and plays a role in resistance to cancer therapies. AIM: In this study, we examined radiation-induced DNA double strand breaks (DSB) in lung cancer cell lines and examined whether La/SSB associated with these DSB. METHOD: Three lung cancer lines (A549, H460 and LL2) were irradiated with different X-ray doses or X-radiated with a 5 Gy dose and examined at different time-points post-irradiation for DNA DSB in the form of γ-H2AX and Rad51 foci. Using fluorescence microscopy, we examined whether La/SSB and γ-H2AX co-localise and performed proximity ligation assay (PLA) and co-immunoprecipitation to confirm the interaction of these proteins. RESULTS: We found that the radio-resistant A549 cell line compared to the radio-sensitive H460 cell line showed faster resolution of radiation-induced γ-H2AX foci over time. Conversely, we found more co-localised γ-H2AX and La/SSB foci by PLA in irradiated A549 cells. CONCLUSION: The co-localisation of La/SSB with radiation-induced DNA breaks suggests a role of La/SSB in DNA repair, however further experimentation is required to validate this.

Characterization of Sequence-Specific Binding of LARP6 to the 5' Stem-Loop of Type I Collagen mRNAs and Implications for Rational Design of Antifibrotic Drugs.

Excessive synthesis of type I collagen is a hallmark of fibrotic diseases. Binding of La-related protein 6 (LARP6) to the 5' stem-loop (5'SL) of collagen mRNAs regulates their translation leading to an unnaturally elevated rate of collagen biosynthesis in fibrosis. Previous work suggested that LARP6 needs two domains to form stable complex with 5'SL RNA, the La domain and the juxtaposed RNA recognition motif (RRM), jointly called the La-module. Here we describe that La domain of LARP6 is necessary and sufficient for recognition of 5'SL in RNA sequence specific manner. A three-amino-acid motif located in the flexible loop connecting the second α-helix to the ß-sheet of the La domain, called the RNK-motif, is critical for binding. Mutation of any of these three amino acids abolishes the binding of the La domain to 5'SL. The major site of crosslinking of LARP6 to 5'SL RNA was mapped to this motif, as well. The RNK-motif is not found in other LARPs, which cannot bind 5'SL. Presence of RRM increases the stability of complex between La domain and 5'SL RNA and RRM domain does not make extensive contacts with 5'SL RNA. We propose a model in which the initial recognition of 5'SL by LARP6 is mediated by the RNK epitope and further stabilized by the RRM domain. This discovery suggests that the interaction between LARP6 and collagen mRNAs can be blocked by small molecules that target the RNK epitope and will help rational design of the LARP6 binding inhibitors as specific antifibrotic drugs.

LARP6 proteins in plants.

RNA binding proteins, through control of mRNA fate and expression, are key players of organism development. The LARP family of RBPs sharing the La motif, are largely present in eukaryotes. They classify into five subfamilies which members acquired specific additional domains, including the RRM1 moiety which teams up with the La motif to form a versatile RNA binding unit. The LARP6 subfamily has had a peculiar history during plant evolution. While containing a single LARP6 in algae and non-vascular plants, they expanded and neofunctionalized into three subclusters in vascular plants. Studies from Arabidopsis thaliana, support that they acquired specific RNA binding properties and physiological roles. In particular LARP6C participates, through spatiotemporal control of translation, to male fertilization, a role seemingly conserved in maize. Interestingly, human LARP6 also acts in translation control and mRNA transport and similarly to LARP6C which is required for pollen tube guided elongation, is necessary to cell migration, through protrusion extension. This opens the possibility that some cellular and molecular functions of LARP6 were retained across eukaryote evolution. With their peculiar evolutionary history, plants provide a unique opportunity to uncover how La-module RNA binding properties evolved and identify species specific and basal roles of the LARP6 function. Deciphering of how LARP6, in particular LARP6C, acts at the molecular level, will foster novel knowledge on translation regulation and dynamics in changing cellular contexts. Considering the seemingly conserved function of LARP6C in male reproduction, it should fuel studies aimed at deriving crop species with improved seed yields.