La-related Protein Research Articles

Global analysis of LARP1 translation targets reveals tunable and dynamic features of 5′ TOP motifs

Terminal oligopyrimidine (TOP) motifs are sequences at the 5' ends of mRNAs that link their translation to the mTOR Complex 1 (mTORC1) nutrient-sensing signaling pathway. They are commonly regarded as discrete elements that reside on ∼100 mRNAs that mostly encode translation factors. However, the full spectrum of TOP sequences and their prevalence throughout the transcriptome remain unclear, primarily because of uncertainty over the mechanism that detects them. Here, we globally analyzed translation targets of La-related protein 1 (LARP1), an RNA-binding protein and mTORC1 effector that has been shown to repress TOP mRNA translation in a few specific cases. We establish that LARP1 is the primary translation regulator of mRNAs with classical TOP motifs genome-wide, and also that these motifs are extreme instances of a broader continuum of regulatory sequences. We identify the features of TOP sequences that determine their potency and quantify these as a metric that accurately predicts mTORC1/LARP1 regulation called a TOPscore. Analysis of TOPscores across the transcriptomes of 16 mammalian tissues defines a constitutive "core" set of TOP mRNAs, but also identifies tissue-specific TOP mRNAs produced via alternative transcription initiation sites. These results establish the central role of LARP1 in TOP mRNA regulation on a transcriptome scale and show how it connects mTORC1 to a tunable and dynamic program of gene expression that is tailored to specific biological contexts.

The Alazami Syndrome-Associated Protein LARP7 Guides U6 Small Nuclear RNA Modification and Contributes to Splicing Robustness.

The La-related protein 7 (LARP7) forms a complex with the nuclear 7SK RNA to regulate RNA polymerase II transcription. It has been implicated in cancer and the Alazami syndrome, a severe developmental disorder. Here, we report a so far unknown role of this protein in RNA modification. We show that LARP7 physically connects the spliceosomal U6 small nuclear RNA (snRNA) with a distinct subset of box C/D small nucleolar RNAs (snoRNAs) guiding U6 2'-O-methylation. Consistently, these modifications are severely compromised in the absence of LARP7. Although general splicing remains largely unaffected, transcriptome-wide analysis revealed perturbations in alternative splicing in LARP7-depleted cells. Importantly, we identified defects in 2'-O-methylation of the U6 snRNA in Alazami syndrome siblings carrying a LARP7 mutation. Our data identify LARP7 as a bridging factor for snoRNA-guided modification of the U6 snRNA and suggest that alterations in splicing fidelity contribute to the etiology of the Alazami syndrome.

LARP7-Mediated U6 snRNA Modification Ensures Splicing Fidelity and Spermatogenesis in Mice.

U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.

Structural Basis of 7SK RNA 5′-Gamma-Phosphate Methylation and Retention by MePCE

7SK non-coding RNA is an essential regulator of eukaryotic transcription elongation through dynamic RNP assemblies that sequester or release the positive transcription elongation factor b (P-TEFb). All 7SK RNPs include a core comprising 7SK RNA, methylphosphate capping enzyme (MePCE), and La-related protein 7 (Larp7). In addition to its non-enzymatic role as a core component of 7SK RNPs, MePCE catalyzes the formation of a gamma-phosphate monomethylation cap, unique to 7SK and U6 in humans. How MePCE can specifically recognize and modify its RNA substrate and remain bound to 7SK to form the core 7SK RNP has not been previously known. We report 2.0 and 2.1 Å X-ray crystal structures of human MePCE methyltransferase domain bound to S-adenosylhomocysteine (SAH) and uncapped or capped 7SK substrates, respectively. We find that 7SK recognition is achieved by an extensive network of protein contacts to a 5' hairpin-single-stranded RNA region, involving the triphosphate group, two terminal basepairs and six single-stranded nucleotides, thus explaining MePCE specificity for 7SK and U6. The large size of the RNA binding site required for recognition and catalysis is unique among RNA methyltransferases studied to date. The structures reveal SAH and product RNA in a near-transition state geometry, where the cofactor SAH is buried inside the active site by the RNA-interacting residues. Unexpectedly, binding experiments show that MePCE has higher affinity for capped vs uncapped 7SK, with kinetic data supporting a slow product release model. Taken together, these data describe the features of an unusual RNA capping enzyme, and reveal the molecular mechanism of methyl transfer and 7SK retention by MePCE for subsequent assembly of the core 7SK RNP.

Altered platelet proteome in lupus anticoagulant (LA)-positive patients\u2014protein disulfide isomerase and NETosis as new players in LA-related thrombosis

Patients with antiphospholipid syndrome (APS) are at high risk of developing venous and arterial thromboembolism (TE). The role of platelets in the pathogenesis of these prothrombotic conditions is not yet fully understood. The aim of this study was to gain mechanistic insights into the role of platelets in APS by comparing the platelet proteome between lupus anticoagulant (LA)-positive patients with (LA+ TE+) and without a history of TE (LA+ TE−) and healthy controls. The platelet proteome of 47 patients with LA, 31 with a history of TE and 16 without thrombotic history, and 47 healthy controls was analyzed by two-dimensional differential in-gel electrophoresis and mass spectrometry to identify disease-related proteins. Afterward, selected LA-related platelet proteins were validated by western blot and ELISA. Alterations of 25 proteins were observed between the study groups. STRING pathway analysis showed that LA-related protein profiles were involved in platelet activation, aggregation, and degranulation. For example, protein disulfide isomerase family members, enzymes that promote thrombosis, were upregulated in platelets and plasma of LA+ TE+ patients. Leukocyte elastase inhibitor (SERPINB1), an antagonist of neutrophil extracellular trap (NET) formation, was decreased in platelets of LA+ TE+ patients compared to healthy controls. Additionally, citrullinated histone H3, a NET-specific marker, was increased in plasma of LA+ TE+ patients. These findings suggest that decreased platelet SERPINB1 levels favor prothrombotic NETosis, especially in LA+ TE+ patients. Our findings reveal protein abundance changes connected to altered platelet function in LA-positive patients, thus suggesting a pathogenic role of platelets in thrombotic complications in APS.

Subcellular mRNA Localization Regulates Ribosome Biogenesis In Migrating Cells

Translation of Ribosomal Protein-coding mRNAs (RP-mRNAs) constitutes a key step in ribosome biogenesis, but the mechanisms which modulate RP-mRNA translation in coordination with other cellular processes are poorly defined. Here we show that the subcellular localization of RP-mRNAs acts as a key regulator of their translation during cell migration. As cells migrate into their surroundings, RP-mRNAs localize to the actin-rich protrusive fronts of the cells. This localization is mediated by La-related protein 6 (LARP6), an RNA binding protein that is enriched in protrusions. Protrusions act as hotspots of translation for RP-mRNAs, resulting in enhancement of RP synthesis, ribosome biogenesis, and overall protein synthetic capacity of migratory cells, required for supporting efficient migration and proliferation. In human breast carcinomas, Epithelial to Mesenchymal Transition (EMT) upregulates LARP6 expression to enhance protein synthesis and support invasive growth. Our findings reveal LARP6 mediated mRNA localization as a key regulator of ribosome biogenesis during cell migration, and demonstrate a role for this process in cancer progression downstream of EMT.

Use of tRNA-Mediated Suppression to Assess RNA Chaperone Function.

La proteins have well-established roles in the maturation of RNA polymerase III transcripts, including pre-tRNAs. In addition to protecting the 3' end of pre-tRNAs from exonuclease digestion, La proteins also promote the native fold of the pre-tRNA using RNA chaperone activity. tRNA-mediated suppression in the fission yeast S. pombe has been an invaluable tool in determining the mechanistic basis by which La proteins promote the maturation of defective pre-tRNAs that benefit from RNA chaperone activity. More recently, tRNA-mediated suppression has been adapted to test for RNA chaperone function in the La-related proteins and in the promoting of tRNA function by tRNA modification enzymes. Thus tRNA-mediated suppression can be a useful assay for the investigation of various proteins hypothesized to promote tRNA folding through RNA chaperone related activities.

Capturing the Mechanism Underlying TOP mRNA Binding to LARP1

The RNA-binding protein La-related protein 1 (LARP1) plays a central role in ribosome biosynthesis. Its C-terminal DM15 region binds the 7-methylguanosine (m7G) cap and 5' terminal oligopyrimidine (TOP) motif characteristic of transcripts encoding ribosomal proteins and translation factors. Under the control of mammalian target of rapamycin complex 1 (mTORC1), LARP1 regulates translation of these transcripts. Characterizing the dynamics of DM15-TOP recognition is essential to understanding this fundamental biological process. We use molecular dynamics simulations, biophysical assays, and X-ray crystallography to reveal the mechanism of DM15 binding to TOP transcripts. Residues C-terminal to the m7G-binding site play important roles in cap recognition. Furthermore, we show that the unusually static pocket that recognizes the+1 cytosine characteristic of TOP transcripts drives binding specificity. Finally, we demonstrate that the DM15 pockets involved in TOP-specific m7GpppC-motif recognition are likely druggable. Collectively, these studies suggest unique opportunities for further pharmacological development.

The LARP1 La-Module recognizes both ends of TOP mRNAs

ABSTRACT La-Related Protein 1 (LARP1) is an RNA-binding protein that regulates the stability and translation of mRNAs encoding the translation machinery, including ribosomal proteins and translation factors. These mRNAs are characterized by a 5ʹ-terminal oligopyrimidine (TOP) motif that coordinates their temporal and stoichiometric expression. While LARP1 represses TOP mRNA translation via the C-terminal DM15 region, the role of the N-terminal La-Module in the recognition and translational regulation of TOP mRNAs remains elusive. Herein we show that the LARP1 La-Module also binds TOP motifs, although in a cap-independent manner. We also demonstrate that it recognizes poly(A) RNA. Further, our data reveal that the LARP1 La-Module can simultaneously engage TOP motifs and poly(A) RNA. These results evoke an intriguing molecular mechanism whereby LARP1 could regulate translation and stabilization of TOP transcripts.

Mechanisms of Nuclear Export of the RNA‐Binding Protein LARP6

The La Related Protein (LARP) superfamily comprises RNA binding proteins that have diverse RNA targets (including mRNA and tRNA) and exert regulatory functions in the cell. One member of the family, LARP6, regulates the synthesis of type I collagen by binding the 5′ untranslated region of the mRNA and facilitating its localization to the ER membrane. Aberrant collagen expression is a key feature of fibroproliferative diseases such as osteogenesis imperfecta. Therefore, LARP6 is a potential target for treatment of these diseases. However, the mechanisms of LARP6 cellular localization remain unclear. Previously published work transfected a GFP‐tagged LARP6 harboring a deletion of a putative nuclear export sequence (NES‐RBD) within the RNA binding domain. The NES deletion mutant was observed to accumulate in the nucleus, suggesting nucleocytoplasmic shuttling. The purpose of the current study is to understand this nuclear export mechanism in concert with recent structural data for the RNA‐binding domain of LARP6. First, we investigated the localization of endogenous LARP6 in immortalized cell lines using immunocytochemistry and leptomycin B treatment to inhibit classical nuclear transport mechanisms. These experiments demonstrated challenges with the selectivity of commercial antibodies for endogenous levels of human LARP6 in mammalian cells. Additionally, we used a recently‐developed algorithm, LocNES, to identify a second candidate NES motif in the C‐terminus of LARP6 (NES‐CTD) and to clarify the boundaries of the NES‐RBD effectively breaking it into two candidates (NES‐RBD1 and NES‐RBD2). Two approaches were used to test these new candidate motifs. First, the predicted NES motifs were cloned as fusions to GST and purified in preparation for pulldown assays to directly assess interaction with the transporter CRM1. Second, bacterial expression constructs of the wildtype and deletion mutants of the two predicted NES were constructed to analyze the effects of the NES deletions on recombinant protein stability. Chemical and thermal denaturation of the recombinant proteins was monitored by fluorescence spectroscopy to measure the free energy of folding. While all of the GST‐NES fusions are readily expressed in bacteria, only the NES‐CTD candidate motif is readily purified. The high hydrophobicity of the two NES‐RBD candidate motifs appears to prevent their purification. Together, the observed differences in the stability of GST‐NES peptides and the LARP6‐NES deletion mutants suggest that manipulation of the hydrophobic‐rich regions complicates the biochemical efforts to elucidate the mechanism of nuclear export of LARP6.Support or Funding InformationNational Institutes of Health – National Institute of General Medical Sciences (GM119096)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LARP4A recognizes polyA RNA via a novel binding mechanism mediated by disordered regions and involving the PAM2w motif, revealing interplay between PABP, LARP4A and mRNA.

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A–oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein–protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.

Capturing the Mechanism Underlying Top-Binding to the LARP1 DM15 Region

The RNA-binding protein La-related protein 1 (LARP1) plays a central role in the mTORC1 signaling pathway, which regulates the biosynthesis of ribosomes. The LARP1 DM15 region binds the 7-methylguanosine (m7G) cap and terminal oligopyrimidine (TOP) motif of mRNA transcripts that encode ribosomal proteins and translation initiation factors. LARP1 thereby regulates whether these transcripts are available for translation. We describe molecular dynamics simulations and biochemical/biophysical assays that reveal the underlying mechanisms of the DM15/mRNA interaction. Importantly, the simulations reveal that residues C-terminal to the cap-binding pocket play important roles in the dynamics of cap-recognition. The data also reveal an unusually static pocket that recognizes the +1 cytosine that is characteristic of TOP transcripts. We show that this pocket drives the specificity of target binding. Computational hotspot analysis of the DM15 region suggests that these cap- and +1C-binding pockets are indeed druggable. LARP1 is unique in its ability to directly recognize the m7GpppC motif of TOP transcripts. Small-molecule LARP1 ligands that impede DM15/mRNA binding could be developed into treatments for several cancers known to have elevated LARP1 levels.

Abstract B155: Impact of transcription initation on translation regulation by the mTOR pathway

Abstract The two major processes of eukaryotic gene expression, transcription and translation, are spatially and temporally separated and commonly perceived to be independently regulated. Unexpectedly, the analysis of nascent transcription start sites (TSSs) genome-wide, proposes that alternative TSS can target mRNAs of the same gene to distinct modes of translational regulation.Our preliminary data found the TCT core promoter motif to function as a transcriptionally suboptimal Initiator (Inr) motif. Despite the rapid turnover of most regulatory elements, the TCT motif is remarkably conserved among the promoters of orthologous bilaterians genes. However, the translation of mRNAs starting from TCT but not Inr promoters is preferentially regulated by the Mammalian target of rapamycin complex 1 (mTORC1), likely via the La-related protein 1 (Larp1) that binds mRNAs starting on pyrimidine but not purines. These findings reveal a functional dependency between transcription initiation and translation regulation, and highlight the importance of TSS selection in promoters. Inhibitors of mTORC1, and more recently Larp1, are rapidly moving into clinical cancer therapy trials but did not live up to the expectations in patients. Markers for patient stratification and predicting mTOR inhibitor efficacy are needed. As TSSs reflect the integrated signals of gene regulatory pathways and also appear to impact translational regulation, our future research will address how far alternative TSSs facilitate acquired resistance to mTOR inhibitors in tumors and 2) TSSs can serve as a predictive marker for mTOR inhibitors efficacy. Citation Format: Sascha H. Duttke, Max Chang, Christopher K. Glass, Andrea Berman, Christopher Benner. Impact of transcription initation on translation regulation by the mTOR pathway [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B155.

Resonance assignment of human LARP4A La module

Human LARP4A belongs to a superfamily of RNA binding proteins called La-related proteins (LARPs). Whilst being a positive regulator of protein synthesis and a promoter of mRNA stability, LARP4A also controls cell morphology and motility in human breast and prostate cancer cells. All LARPs share a characteristic RNA binding unit named the La–module, which despite a high level of primary structure conservation exhibits a great versatility in RNA target selection. Human LARP4A La–module is the most divergent compared with other LARPs and its RNA recognition properties have only recently started to be revealed. Given the key role of LARP4A protein in cancer cell biology, we have initiated a complete NMR characterisation of its La-module and here we report the assignment of 1H, 15N and 13C resonances resulting from our studies.

La-Related Protein 4 as a Suppressor for Motility of Ovarian Cancer Cells.

The La-related proteins (LARPs) are a family of RNA binding proteins that control the degradation and stabilization of RNAs. As emerging research reveals the biology of each LARP, it is evident that LARPs are dysregulated in some types of cancer. Upregulation of cell motility potentiates the metastatic potential of ovarian cancer cells; however, the roles of LARPs in cell motility remain unknown. In the present study, we investigated the roles of LARPs in the progression of ovarian cancer using SKOV3 human ovarian cancer cells and a public database that integrates microarray-based gene expression data and clinical data. To explore the involvement of LARPs in the cell motility, we performed RNA interference screening for LARP mRNAs in SKOV3 cells. The screening identified LARP4 as a potential suppressor of the formation of lamellipodia. Conversely, enforced expression of LARP4 suppressed the formation of lamellipodia. Moreover, cell migration was significantly increased in LARP4-depleted SKOV3 cells. Mechanistically, LARP4 depletion was associated with the decrease in RhoA protein expression. These results suggest that LARP4 may limit RhoA-dependent cell motility. In a mouse xenograft model with SKOV3 cells, LARP4 depletion potentiated peritoneal metastasis. Upon analysis of a public database of patients with ovarian cancer, the LARP4 mRNA-high expression group (n = 166) showed longer overall survival compared with the LARP4 mRNA-low expression group (n = 489), implying a positive correlation of LARP4 mRNA levels in ovarian cancer tissues with patient prognosis. Taken together, we propose that LARP4 could suppress motility and metastatic potential of ovarian cancer cells.

Identification of salt stress response genes using the Artemia transcriptome

Habitat salinity is a major abiotic factor governing the activity, physiology, biology and distribution of aquatic animals. Salinity changes cause salt stress, affecting crustaceans reared in aquaculture both on an ecological and economic level. Current salt stress research in aquatic animals is mainly focused on salt stress in the gills at relatively low salinity ranges. Knowledge about whole-body salinity response in crustaceans and other organisms is lacking, especially in hypersaline conditions.Artemia franciscana is a small halophilic model crustacean able to withstand high salinities up to 300 g/l and strong osmotic shocks thanks to its mitigating strategies for fluctuating salinity levels, such as its unique larval salt gland and osmoregulatory capacity. This study aims to identify the genes responsible for Artemia's unique hypersalinity tolerance by differential expression analysis.First, the full transcriptome of A. franciscana in different metabolic and life cycle stages was assembled de novo (assembly statistics: N50 = 1,430; GC content = 35.63%; transcript number = 64,972) and functionally annotated (annotated transcripts = 36%). Then, naupliar RNA-Seq reads generated under respectively hypersaline and marine conditions were pseudo-aligned to the A. franciscana transcriptome. Expression levels in both conditions were finally compared and 177 differentially expressed, functionally annotated transcripts were identified, of which 113 transcripts with GO annotations. Signalling genes, such as EIF and several genes from the glutathione and the chitin metabolic pathways were induced in Artemia under hypersaline conditions. Hypersalinity also activated gene regulation mechanisms (expression, transcription and post transcription) in the nucleus for DNA repair, ubiquitination, and also for cell cycle arrest through La-related protein. Several lipid metabolic genes and lipid transporters were upregulated, potentially to provide energy for ion balance and to maintain membrane structure integrity. Transport of metal ions and other ions was upregulated as well to maintain ion homeostasis. Lastly, known crustacean stress response genes such as Heat shock 70 kDa protein cognate were upregulated. This work shows that salt stress in Artemia nauplii, through signal transduction, gene regulation, lipid metabolism, transport and stress response genes, has an important influence on known and novel homeostasis-repairing mechanisms in Artemia.

Structural basis for recognition of human 7SK long noncoding RNA by the La-related protein Larp7

The La and the La-related protein (LARP) superfamily is a diverse class of RNA binding proteins involved in RNA processing, folding, and function. Larp7 binds to the abundant long noncoding 7SK RNA and is required for 7SK ribonucleoprotein (RNP) assembly and function. The 7SK RNP sequesters a pool of the positive transcription elongation factor b (P-TEFb) in an inactive state; on release, P-TEFb phosphorylates RNA Polymerase II to stimulate transcription elongation. Despite its essential role in transcription, limited structural information is available for the 7SK RNP, particularly for protein-RNA interactions. Larp7 contains an N-terminal La module that binds UUU-3'OH and a C-terminal atypical RNA recognition motif (xRRM) required for specific binding to 7SK and P-TEFb assembly. Deletion of the xRRM is linked to gastric cancer in humans. We report the 2.2-Å X-ray crystal structure of the human La-related protein group 7 (hLarp7) xRRM bound to the 7SK stem-loop 4, revealing a unique binding interface. Contributions of observed interactions to binding affinity were investigated by mutagenesis and isothermal titration calorimetry. NMR 13C spin relaxation data and comparison of free xRRM, RNA, and xRRM-RNA structures show that the xRRM is preordered to bind a flexible loop 4. Combining structures of the hLarp7 La module and the xRRM-7SK complex presented here, we propose a structural model for Larp7 binding to the 7SK 3' end and mechanism for 7SK RNP assembly. This work provides insight into how this domain contributes to 7SK recognition and assembly of the core 7SK RNP.

LARP1 on TOP of ribosome production.

The ribosome is an essential unit of all living organisms that commands protein synthesis, ultimately fuelling cell growth (accumulation of cell mass) and cell proliferation (increase in cell number). The eukaryotic ribosome consists of 4 ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs). Despite its fundamental role in every living organism, our present understanding of how higher eukaryotes produce the various ribosome components is incomplete. Uncovering the mechanisms utilized by human cells to generate functional ribosomes will likely have far-reaching implications in human disease. Recent biochemical and structural studies revealed La-related protein 1 (LARP1) as a key new player in RP production. LARP1 is an RNA-binding protein that belongs to the LARP superfamily; it controls the translation and stability of the mRNAs that encode RPs and translation factors, which are characterized by a 5' terminal oligopyrimidine (5'TOP) motif and are thus known as TOP mRNAs. The activity of LARP1 is regulated by the mammalian target of rapamycin complex 1 (mTORC1): a eukaryotic protein kinase complex that integrates nutrient sensing with mRNA translation, particularly that of TOP mRNAs. In this review, we provide an overview of the role of LARP1 in the control of ribosome production in multicellular eukaryotes. This article is categorized under: Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Processing > Capping and 5' End Modifications.

A structural role for the C‐terminal conserved LSA motifs in Arabidopsis LARP6 proteins

La Related Protein 6 (LARP6) is a sub‐family of the RNA‐binding La‐Related Proteins superfamily (LARPs). In vertebrates, LARP6 is known to bind to the 5′ untranslated region of mRNAs that encode for collagen type I. In contrast, the ligands and biological function of the three LARP6 paralogs in plant LARP6 (LARP6a, LARP6b and LARP6c) remain to be determined. Of the three variations of the LARP6 protein in Arabidopsis thaliana, LARP6b and LARP6c contain a PAM2 motif in the N terminal region of the protein, while LARP6a does not. All three LARP6 paralogs contain the conserved LSA motif, which is a distinguishing feature of the LARP6 subfamily and has been implicated in biologically relevant protein‐protein interactions. We have successfully expressed and purified full‐length LARP6a, LARP6b, and LARP6c proteins from Arabidopsis thaliana as recombinant proteins in E. coli. Mutant forms of the Arabidopsis LARP6 proteins were generated using site‐directed mutagenesis to test the role of conserved sequence motifs. The deletion of the PAM2 motif from LARP6c does not negatively impact the structural stability of the full‐length protein. However, deletion of the LSA from LARP6c does appear to destabilize the recombinant protein during biosynthesis. We hypothesize that the LSA motif in plant LARP6 proteins contributes to the global structure and/or the thermodynamic stability of the proteins. These studies using highly purified recombinant protein, with protein stability measured by differential scanning calorimetry, provide novel insight into the potential structure and function of the uncharacterized LSA motif that characterizes the LARP6 subfamily of RNA‐binding proteins.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Major Boundaries for Structural Characterization of the LARP6 Proteins From Fish

La‐related protein 6 (LARP6) is a posttranscriptional regulator of collagen type I expression in vertebrates. It binds a conserved stem loop structure in the untranslated region of the COL1A1 mRNA. LARP6 contains an uncharacterized N‐terminal domain, a centrally‐located RNA binding domain, and an uncharacterized C‐terminal domain that includes a proline‐rich motif called the LSA motif. The human LARP6 La Module has been structurally characterized as two subdomains: a winged‐helix La Motif (LaM) and an RNA recognition motif (RRM). Both domains are necessary to carry out the RNA binding activity. Previous work in the lab identified that the LARP6 proteins from two bony fish, Danio rerio and Xiphophorus maculatus, contain a proteolytically‐resistant structure that is comprised of both the La Module and the uncharacterized N‐terminal domain. To better understand the interactions between these two components, we set out to precisely identify the domain boundaries of the N‐terminal domain (NTD), La Module (LaMod), and C‐terminal domain (CTD) in the fish LARP6 proteins. We predicted domain boundaries based on multiple sequence alignments, and cloned those boundaries into bacterial expression vectors to create constructs for the isolated La Module and a deletion of just the C‐terminal domain (DCTD = NTD+LaMod) for Homo sapiens, Danio rerio, and Xiphophorus maculatus LARP6. All six of these proteins have been recombinantly expressed and purified using immobilized affinity and size exclusion chromatography. The purified proteins were screened for proper folding by analyzing RNA binding activity using electrophoretic mobility shift assay with the HsCOL1A1 RNA ligand. Proteins were also analyzed for structural stability using limited proteolysis with either trypsin or aminopeptidase. The proteolysis reactions were evaluated by SDS‐PAGE and silver stain, and the major bands excised and further analyzed by mass spectrometry. By mapping the recovered proteolytic peptides onto the protein sequence, we were able to identify conservative boundaries for each of the constructs, which were then tested by the construction, purification, and biochemical analysis of new recombinant protein constructs. Having precise boundaries for the stable domains within the fish LARP6 proteins will enable the structural characterization of these orthologs for comparison to the human protein. This future work will provide insight into the variation of structure and function of these highly‐conserved post‐transcriptional regulators.Support or Funding InformationFunding provided by the Texas State University Student Undergraduate Research Fund (to E.L.P.), the National Institutes of Health – National Institute of General Medical Studies (GM119096 to K.A.L; GM102783, the South Texas Doctoral Bridge Program, supporting J.M.C., E.S. & E.L.P., and GM107759).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.