La-related Protein Research Articles

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.

Cellular RNA-binding proteins LARP4 and PABPC1 synergistically facilitate viral translation of coronavirus PEDV

Coronaviruses are causing epizootic diseases and thus are a substantial threat for both domestic and wild animals. These viruses depend on the host translation machinery to complete their life cycle. The current paper identified cellular RNA-binding proteins (RBPs), La-related protein 4 (LARP4) and polyadenylate-binding protein cytoplasmic 1 (PABPC1), as critical regulators of efficient translation of the coronavirus porcine epidemic diarrhea virus (PEDV) mRNA. In Vero cells, PEDV infection caused LARP4 to migrate from the nucleus to the cytoplasm in a chromosome region maintenance1 (CRM1)-independent pathway. In the absence of the nuclear export signal of LARP4, viral translation was not promoted by LARP4. A further study unveiled that the cytoplasmic LARP4 binds to the 3′-terminal untranslated region (3′UTR) of PEDV mRNA with the assistance of PABPC1 to facilitate viral translation. LARP4 knockdown reduced the promotion of the PABPC1-induced 3′UTR translation activity. Moreover, the rabbit reticulocyte lysate (RRL) system revealed that the prokaryotic expressed protein LARP4 and PABPC1 enhance PEDV mRNA translation. To our knowledge, this is the first study demonstrating that PEDV induces nucleo-cytoplasmic shuttling of LARP4 to enhance its own replication, which broadens our insights into how viruses use host's RBPs for the efficient translation of viral mRNA.

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

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. 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. 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. Targeted inhibition of LARP7 or STING expression may be a potential therapeutic strategy for the treatment of DCM.

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

ABSTRACT 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.

Deciphering the Binding of 5' Stem Loop RNA to the La Domain of Human LARP6.

La-related protein 6 regulates the highly organized biosynthesis of type I procollagen polypeptides and affects proper assembly of procollagen peptides into heterotrimers of type I procollagen. LARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5' stem loop motif found in type I and III collagen mRNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic instability of the La domain of HsLARP6 hampers studies probing the molecular basis of biologically- and disease-relevant structure-function relationship, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effect of the construct length as well as RNA binding on protein stability. N- and C-terminal extensions greatly impact stability based on interactions with the core domain and modulation of the pI. When in complex with its cognate 5'SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50x longer than the unbound state, under identical conditions, likely due to a global change in conformational plasticity upon RNA binding. These results provide a foundation for further studies of the molecular recognition of 5'SL by HsLARP6 as well as a platform for refining potential antifibrotic therapeutics.

EIF4A1 enhances LARP1-mediated translational repression during mTORC1 inhibition.

Eukaryotic translation initiation factor (eIF)4A-a DEAD-box RNA-binding protein-plays an essential role in translation initiation. Recent reports have suggested helicase-dependent and helicase-independent functions for eIF4A, but the multifaceted roles of eIF4A have not been fully explored. Here we show that eIF4A1 enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA pulldown followed by sequencing revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTORC1 inhibition. This selective interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that deletion of EIF4A1 attenuated the translational repression of TOP mRNAs upon mTORC1 inactivation. Moreover, eIF4A1 increases the interaction between TOP mRNAs and LARP1 and, thus, ensures stronger translational repression upon mTORC1 inhibition. Our data show the multimodality of eIF4A1 in modulating protein synthesis through an inhibitory binding partner and provide a unique example of the repressive role of a universal translational activator.

LARP4B promotes hepatocellular carcinoma progression and impairs sorafenib efficacy by activating SPINK1-mediated EGFR pathway.

La-related proteins (LARPs) regulate gene expression by binding to RNAs and exhibit critical effects on disease progression, including tumors. However, the role of LARP4B and its underlying mechanisms in the progression of hepatocellular carcinoma (HCC) remain largely unclear. In this study, we found that LARP4B expression is upregulated and correlates with poor prognosis in patients with HCC. Gain- and loss-of-function assays showed that LARP4B promotes stemness, proliferation, metastasis, and angiogenesis in vitro and in vivo. Furthermore, LARP4B inhibition enhances the antitumor effects of sorafenib and blocks the metastasis-enhancing effects of low sorafenib concentrations in HCC. Mechanistically, LARP4B expression is upregulated by METTL3-mediated N6-methyladenosine (m6A)-IGF2BP3-dependent modification in HCC. RNA- and RNA immunoprecipitation (RIP)- sequencing uncovered that LARP4B upregulates SPINK1 by binding to SPINK1 mRNA via the La motif and maintaining mRNA stability. LARP4B activates the SPINK1-mediated EGFR signaling pathway, which supports stemness, progression and sorafenib resistance in HCC. Additionally, a positive feedback loop with the LARP4B/SPINK1/p-AKT/C/EBP-β axis is responsible for the sorafenib-therapeutic benefit of LARP4B depletion. Overall, this study demonstrated that LARP4B facilitates HCC progression, and LARP4B inhibition provides benefits to sorafenib treatment in HCC, suggesting that LARP4B might be a potential therapeutic target for HCC.

1H, 13C, and 15N resonance assignments of the La Motif of the human La-related protein 1.

Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5'terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.

Genome-wide investigation of the LARP gene family: focus on functional identification and transcriptome profiling of ZmLARP6c1 in maize pollen

BackgroundThe La-related proteins (LARPs) are a superfamily of RNA-binding proteins associated with regulation of gene expression. Evidence points to an important role for post-transcriptional control of gene expression in germinating pollen tubes, which could be aided by RNA-binding proteins.ResultsIn this study, a genome-wide investigation of the LARP proteins in eight plant species was performed. The LARP proteins were classified into three families based on a phylogenetic analysis. The gene structure, conserved motifs, cis-acting elements in the promoter, and gene expression profiles were investigated to provide a comprehensive overview of the evolutionary history and potential functions of ZmLARP genes in maize. Moreover, ZmLARP6c1 was specifically expressed in pollen and ZmLARP6c1 was localized to the nucleus and cytoplasm in maize protoplasts. Overexpression of ZmLARP6c1 enhanced the percentage pollen germination compared with that of wild-type pollen. In addition, transcriptome profiling analysis revealed that differentially expressed genes included PABP homologous genes and genes involved in jasmonic acid and abscisic acid biosynthesis, metabolism, signaling pathways and response in a Zmlarp6c1::Ds mutant and ZmLARP6c1-overexpression line compared with the corresponding wild type.ConclusionsThe findings provide a basis for further evolutionary and functional analyses, and provide insight into the critical regulatory function of ZmLARP6c1 in maize pollen germination.

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.

Distinct roles of LARP1 and 4EBP1/2 in regulating translation and stability of 5'TOP mRNAs.

A central mechanism of mTOR complex 1 (mTORC1) signaling is the coordinated translation of ribosomal protein and translation factor mRNAs mediated by the 5'-terminal oligopyrimidine motif (5'TOP). Recently, La-related protein 1 (LARP1) was proposed to be the specific regulator of 5'TOP mRNA translation downstream of mTORC1, while eIF4E-binding proteins (4EBP1/2) were suggested to have a general role in translational repression of all transcripts. Here, we use single-molecule translation site imaging of 5'TOP and canonical mRNAs to study the translation of single mRNAs in living cells. Our data reveal that 4EBP1/2 has a dominant role in repression of translation of both 5'TOP and canonical mRNAs during pharmacological inhibition of mTOR. In contrast, we find that LARP1 selectively protects 5'TOP mRNAs from degradation in a transcriptome-wide analysis of mRNA half-lives. Our results clarify the roles of 4EBP1/2 and LARP1 in regulating 5'TOP mRNAs and provide a framework to further study how these factors control cell growth during development and disease.

LARP1 knockdown inhibits cultured gastric carcinoma cell cycle progression and metastatic behavior.

This study aimed to clarify the role of la-related protein 1 (LARP1) in cell cycle progression and metastatic behavior of cultured gastric carcinoma (GC) cells. To do that, LARP1 expression was detected in clinical GC tissues and cell lines using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The cell viability, apoptosis, cell cycle, migration, invasion, and cell growth were examined using a Cell Counting Kit-8, Annexin V-FITC staining, propidium iodide staining, Transwell migration and invasion assays, and colony formation assays after LARP1 knockdown. Phosphatidyl inositol 3-kinase (PI3K) and AKT1 mRNA and protein expression levels of PI3K, p-AKT1, AKT1, p-BAD, p-mTOR, and p21 in si-LARP1 transfected GC cells were determined using qRT-PCR and western blotting. Here, we've shown that LARP1 expression was upregulated in human GC tissues and KATO III cells. LARP1 knockdown inhibited GC cell proliferation, cell cycle progression, migration, invasion, and colony formation and promoted apoptosis. In si-LARP1-transfected KATO III cells, the mRNA expression levels of PI3K and AKT1, PI3K protein expression, and the p-AKT1/AKT1 ratio were significantly suppressed. p-mTOR and p-BAD were significantly decreased, whereas p21 was significantly increased in si-LARP1-transfected KATO III cells. In conclusion LARP1 knockdown induces apoptosis and inhibits cell cycle progression and metastatic behavior via PI3K/AKT1 signaling in GC cells.

The La-related protein PsLARP4_5 is crucial for zoospore production and pathogenicity in Phytophthora sojae

A large number of La-related proteins (LARPs), most of which share a La-motif and one or more adjacent RNA-recognition (RRM) domains, are known to play a function in diverse processes. Among the LAPRs, LARP4 and LARP5 have been mainly reported to act as positive translation factors. In Phytophthora sojae, only one protein with typical LARP protein features was identified, and it was named LARP4_5 due to the presence of an RRM_LARP4_5-like domain. The PsLARP4_5 gene was significantly upregulated in zoospores and during the infection stage. By comparing the biological characteristics of a wild-type strain with three PsLARP4_5 knockout transformants, it was found that PsLARP4_5 was involved in mycelial growth, sporangium and zoospore production, and pathogenicity of P. sojae. Further analysis of the transcriptome indicated that many differentially expressed genes could participate in several essential biological processes in the PsLARP4_5 transformant, including translation, as structural constituents of ribosomes or cytosolic large ribosomal subunits and others. Notably, 76 genes with a role in the ribosome pathway were downregulated in the PsLARP4_5 transformant, suggesting that PsLARP4_5 might affect translation. Overall, these findings indicate that PsLARP4_5 plays an essential role in the development and pathogenicity of P. sojae.

Abstract 18465: Regulation of Collagen Synthesis and Fibrosis Development in Skeletal Muscle by La-Related Protein 6 (LaRP6)

Introduction: Excessive extracellular matrix (ECM) accumulation, especially collagens, impairs muscle function in muscular dystrophies, aging and muscle trauma (fibrosis). We have found increased skeletal muscle fibrosis in congestive heart failure (CHF) conditions, likely contributing to the impaired muscle functions. Hypothesis: We hypothesized that LARP6 (La Ribonucleoprotein Domain Family Member 6), an mRNA binding protein that coordinates efficient translation of Collagen I α chains, regulates collagen synthesis during skeletal muscle fibrosis in CHF conditions. Approach: Skeletal muscle fibrosis was analyzed in two mouse models, angiotensin II (Ang II) infusion and left anterior descending artery (LAD) ligation. Expression and subcellular localization of Collagen I, Larp6 and myogenic markers (myogenin and Myh3) were analyzed by qRT-PCR, western blotting, in situ hybridization (RNAScope) and immunostaining in hindlimb muscles in vivo and primary myoblasts and fibro/adipogenic progenitor (FAP) cells in vitro . Collagen I levels were also analyzed by picrosirius red staining (PSR). These analyses were performed in a newly-generated Larp6 knockout (Larp6 -/- ) mice. Results: In both Ang II and LAD ligation models, Collagen I mRNA and protein levels were increased together with LARP6, suggesting that LARP6 may regulate Collagen I synthesis in these conditions. In cultured myoblasts and FAPs, LARP6 protein was co-localized with Col1a1 mRNA at endoplasmic reticulum (ER). TGF-β treatment increased the LARP6 and Col1a1 mRNA co-localization, associated with increased pro-collagen I protein levels. Although Larp6 deficiency did not alter basal Collagen I levels, Collagen I induction in fibrotic conditions was decreased in Larp6 -/- mice. We found that thick collagen fibers were more significantly decreased compared to intermediate or loosely-packed thin collagen fibers (PSR signal analysis under the polarized microscope). Conclusions: These data suggest that LARP6 regulates Collagen I protein synthesis and its filament assembly likely in ER by binding to Col1a1 mRNA upon fibrosis induction. Suppression of LARP6 may be a therapeutic strategy to prevent pathophysiological fibrosis development in skeletal muscle.

The LARP1 homolog Slr1p controls the stability and expression of proto-5′TOP mRNAs in fission yeast

Messenger RNAs (mRNAs) in higher eukaryotes that encode proteins important for the assembly of the translational apparatus (e.g., ribosomal proteins) often harbor a pyrimidine-rich motif at the extreme 5' end known as a 5' terminal oligopyrimidine (5'TOP) sequence. Members of the La-related protein 1 (LARP1) family control 5'TOP expression through a conserved DM15 motif, but the mechanism is not well understood. 5'TOP motifs have not been described in many lower organisms, and fission yeast harbors a LARP1 homolog that also lacks a DM15 motif. In this work, we show that the fission yeast LARP1 homolog, Slr1p, controls the translation and stability of mRNAs encoding proteins analogous to 5'TOP mRNAs in higher eukaryotes, which we thus refer to as proto-5'TOPs. Our data suggest that the LARP1 DM15 motif and the mRNA 5'TOP motif may be features that were scaffolded over a more fundamental mechanism of LARP1-associated control of gene expression.

La-related protein 4 is enriched in vaccinia virus factories and is required for efficient viral replication in primary human fibroblasts.

In addition to the 3'-poly(A) tail, vaccinia virus mRNAs synthesized after viral DNA replication (post-replicative mRNAs) possess a 5'-poly(A) leader that confers a translational advantage in virally infected cells. These mRNAs are synthesized in viral factories, the cytoplasmic compartment where vaccinia virus DNA replication, mRNA synthesis, and translation occur. However, a previous study indicates that the poly(A)-binding protein (PABPC1)-which has a well-established role in RNA stability and translation-is absent in the viral factories. This prompts the question of whether other poly(A)-binding proteins engage vaccinia virus post-replicative mRNA in viral factories. Here, in this study, we found that La-related protein 4 (LARP4), a poly(A) binding protein, was enriched in viral factories in multiple types of cells during vaccinia virus infection. Further studies showed that LARP4 enrichment in the viral factories required viral post-replicative gene expression and functional decapping enzymes encoded by vaccinia virus. We further showed that knockdown of LARP4 expression in human foreskin fibroblasts (HFFs) reduced vaccinia virus DNA replication, post-replicative protein levels, and viral production. Interestingly, the knockdown of LARP4 expression also reduced protein levels from transfected mRNA containing a 5'-poly(A) leader in vaccinia virus-infected and uninfected HFFs. Taken together, our results identified a poly(A)-binding protein, LARP4, being enriched in the vaccinia virus viral factories and facilitating viral replication in HFFs. IMPORTANCE Vaccinia virus, the prototype poxvirus, encodes over 200 open reading frames (ORFs). Over 90 of vaccinia virus ORFs are transcribed post-viral DNA replication. All these mRNAs contain a 5'-poly(A) leader, as well as a 3'-poly(A) tail. They are synthesized in viral factories, where vaccinia virus DNA replication, mRNA synthesis, and translation occur. However, surprisingly, the poly(A) binding protein, PABPC1, that is important for mRNA metabolism and translation is not present in the viral factories, suggesting other poly(A) binding protein(s) may be present in viral factories. Here, we found another poly(A)-binding protein, La-related protein 4 (LARP4), enriched in viral factories during vaccinia virus infection. We also showed that LARP4 enrichment in the viral factories depends on viral post-replicative gene expression and functional viral decapping enzymes. The knockdown of LARP4 expression in human foreskin fibroblasts reduced vaccinia virus DNA replication, post-replicative gene expression, and viral production.

Structure of LARP7 Protein p65–telomerase RNA Complex in Telomerase Revealed by Cryo-EM and NMR

La-related protein 7 (LARP7) are a family of RNA chaperones that protect the 3′-end of RNA and are components of specific ribonucleoprotein complexes (RNP). In Tetrahymena thermophila telomerase, LARP7 protein p65 together with telomerase reverse transcriptase (TERT) and telomerase RNA (TER) form the core RNP. p65 has four known domains—N-terminal domain (NTD), La motif (LaM), RNA recognition motif 1 (RRM1), and C-terminal xRRM2. To date, only the xRRM2 and LaM and their interactions with TER have been structurally characterized. Conformational dynamics leading to low resolution in cryo-EM density maps have limited our understanding of how full-length p65 specifically recognizes and remodels TER for telomerase assembly. Here, we combined focused classification of Tetrahymena telomerase cryo-EM maps with NMR spectroscopy to determine the structure of p65–TER. Three previously unknown helices are identified, one in the otherwise intrinsically disordered NTD that binds the La module, one that extends RRM1, and another preceding xRRM2, that stabilize p65–TER interactions. The extended La module (αN, LaM and RRM1) interacts with the four 3′ terminal U nucleotides, while LaM and αN additionally interact with TER pseudoknot, and LaM with stem 1 and 5′ end. Our results reveal the extensive p65–TER interactions that promote TER 3′-end protection, TER folding, and core RNP assembly and stabilization. The structure of full-length p65 with TER also sheds light on the biological roles of genuine La and LARP7 proteins as RNA chaperones and core RNP components.

Abstract A39: Role of LARP1 in the leukemogenesis of Acute Myeloid Leukemia

Abstract Mammalian target of rapamycin (mTOR) is a strong driver of tumorigenesis in multiple types of tumors, however targeting mTOR for cancer therapy has yielded only limited success. This may be explained in part by signaling redundancy with other pathways, such as CDK9 mTOR-Like (CTORC) complexes, recently described by our group. One of the binders common to mTORC1 and CTORC2 is La related protein 1 (LARP1). LARP1 influences ribosome biogenesis by regulating translation of 5'terminal oligopyrimidine tract (5’ TOP) containing mRNAs, which often encode ribosomal proteins and translation factors. Here, we validate LARP1 as common target of mTORC1 and CTORC2 as well as investigate its influence on leukemogenesis. To explore LARP1’s role in leukemogenesis we utilized CRISPR/CAS9 technology to create OCI-AML5 and U937 LARP1 knockout (KO) cells. Loss of LARP1 expression significantly diminished proliferation potential of AML cell lines both in vitro as well as in vivo when implanted into the flank of athymic nude mice as xenografts. Additionally, primary CD34+ leukemia cells were transfected with LARP1 targeting or scrambled siRNA and leukemic progenitor growth was assessed in methocellulose colony formation assays. We observed suppression of leukemic progenitors colony forming ability in LARP1 siRNA transfected cells, compared to the Ctrl siRNA transfected cells. We also determined enhanced sensitivity of Larp1 KO cell lines to standard chemotherapy agents such Azacitidine, Cytarabine and Venetoclax. We employed polysome profiling to compare global translation levels between Larp1 KO and control U937 cells. Our polysomal profiling results show that KO of LARP1 in U937 cells reduces global translation and ribosome biogenesis as expected as we observed a suppression of both monosomal and polysomal peaks. In summary, Larp1 is important for leukemogenesis and influences their response to treatment with currently used chemotherapy agents. These observations together suggest that LARP1 is a promising target for development of novel antileukemia approaches. Citation Format: Dominik A Nahotko, Aneta H Baran, Mariafausta Fischietti, Elspeth M Beauchamp, Leonidas C Platanias. Role of LARP1 in the leukemogenesis of Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A39.

Interaction of LARP4 to filamin A mechanosensing domain regulates cell migrations.

Filamin A (FLNA) is an actin cross-linking protein that mediates mechanotransduction. Force-dependent conformational changes of FLNA molecule expose cryptic binding site of FLNA, allowing interaction with partners such as integrin, smoothelin, and fimbacin. Here, we identified La-related protein 4 (LARP4) as a new FLNA mechanobinding partner. LARP4 specifically interacts with the cleft formed by C and D strands of immunoglobulin-like repeat 21 (R21) which is blocked by A strand of R20 without force. We validated the interaction between LARP4 and FLNA R21 both in vivo and in vitro. We also determined the critical amino acid that is responsible for the interaction and generated the non-FLNA-binding mutant LARP4 (F277A in human: F273A in mouse Larp4) that disrupts the interaction. Fluorescence recovery after photobleaching (FRAP) of GFP-labeled LARP4 in living cells demonstrated that mutant LARP4 diffuses faster than WT LARP4. Proximity ligation assay (PLA) also confirmed their interaction and disruption of actin polymerization diminishes the interaction. Data mining of RNAseq analysis of LARP4 knockdown (KD) HEK293T cells suggested that LARP4 is involved in morphogenesis and cell motility. Consistent with this prediction, we found that KD of LARP4 increases cell migration speed and expression of the F277A mutant LARP4 in LARP4-KD cells also leads to a higher cell migration speed compared to WT LARP4. These results demonstrated that the LARP4 interaction with FLNA regulates cell migration.

Interaction of the La-related protein Slf1 with colliding ribosomes maintains translation of oxidative-stress responsive mRNAs.

In response to oxidative stress cells reprogram gene expression to enhance levels of antioxidant enzymes and promote survival. In Saccharomyces cerevisiae the polysome-interacting La-related proteins (LARPs) Slf1 and Sro9 aid adaptation of protein synthesis during stress by undetermined means. To gain insight in their mechanisms of action in stress responses, we determined LARP mRNA binding positions in stressed and unstressed cells. Both proteins bind within coding regions of stress-regulated antioxidant enzyme and other highly translated mRNAs in both optimal and stressed conditions. LARP interaction sites are framed and enriched with ribosome footprints suggesting ribosome-LARP-mRNA complexes are identified. Although stress-induced translation of antioxidant enzyme mRNAs is attenuated in slf1Δ, these mRNAs remain on polysomes. Focusing further on Slf1, we find it binds to both monosomes and disomes following RNase treatment. slf1Δ reduces disome enrichment during stress and alters programmed ribosome frameshifting rates. We propose that Slf1 is a ribosome-associated translational modulator that stabilises stalled/collided ribosomes, prevents ribosome frameshifting and so promotes translation of a set of highly-translated mRNAs that together facilitate cell survival and adaptation to stress.