Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADP ribosylation dependent manner to induce translational stalling.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.

Control of mRNA Splicing by Intragenic RNA Activators of Stress Signaling: Potential Implications for Human Disease.

A critical step in the cellular stress response is transient activation of the RNA-dependent protein kinase PKR by double-helical RNA, resulting in down-regulation of protein synthesis through phosphorylation of the α chain of translation initiation factor eIF2, a major PKR substrate. However, intragenic elements of 100-200 nucleotides in length within primary transcripts of cellular genes, exemplified by the tumor necrosis factor (TNF)-α gene and fetal and adult globin genes, are capable of forming RNA structures that potently activate PKR and thereby strongly enhance mRNA splicing efficiency. By inducing nuclear eIF2α phosphorylation, these PKR activator elements enable highly efficient early spliceosome assembly yet do not impair translation of the mature spliced mRNA. The TNF-α RNA activator of PKR folds into a compact pseudoknot that is highly conserved within the phylogeny. Upon excision of ß-globin first intron, the RNA activator of PKR, located in exon 1, is silenced through strand displacement by a short sequence within exon 2, restricting thereby the ability to activate PKR to the splicing process without impeding subsequent synthesis of ß-globin essential for survival. This activator/silencer mechanism likewise controls splicing of α-globin pre-mRNA, but the exonic locations of PKR activator and silencer sequences are reversed, demonstrating evolutionary flexibility. Impaired splicing efficiency may underlie numerous human ß-thalassemia mutations that map to the ß-globin RNA activator of PKR or its silencer. Even where such mutations change the encoded amino acid sequence during subsequent translation, they carry the potential of first impairing PKR-dependent mRNA splicing or shutoff of PKR activation needed for optimal translation.

Control of mRNA splicing by noncoding intragenic RNA elements that evoke a cellular stress response.

Once activated by double-helical RNA, mammalian RNA-dependent stress protein kinase, PKR, phosphorylates its substrate, translation initiation factor eIF2α, to inhibit translation. eIF2α phosphorylation is critical for mounting a cellular stress response. We describe short, 100-200 nucleotide elements within cellular genes that, once transcribed, form RNA structures that potently activate PKR in the vicinity of the RNA and thereby tightly regulate gene expression in cis. Intragenic RNA activators of PKR can (a) attenuate translation of the encoded mRNA by activating PKR and inducing eIF2α phosphorylation, exemplified by the IFN-γ gene, or (b) greatly enhance mRNA splicing efficiency by activating PKR and inducing nuclear eIF2α phosphorylation, thus enabling efficient early spliceosome assembly, exemplified by the adult and fetal globin genes and the TNF-α gene that activates PKR through an RNA pseudoknot conserved from fish to humans. These opposite outcomes considerably extend the potential scope of gene regulation by these novel RNA elements.

PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly.

Short elements in mammalian mRNA can control gene expression by activating the RNA-dependent protein kinase PKR that attenuates translation by phosphorylating cytoplasmic eukaryotic initiation factor 2α (eIF2α). We demonstrate a novel, positive role for PKR activation and eIF2α phosphorylation in human globin mRNA splicing. PKR localizes in splicing complexes and associates with splicing factor SC35. Splicing and early-stage spliceosome assembly on ß-globin pre-mRNA depend strictly on activation of PKR by a codon-containing RNA fragment within exon 1 and on phosphorylation of nuclear eIF2α on Serine 51. Nonphosphorylatable mutant eIF2αS51A blocked ß-globin mRNA splicing in cells and nuclear extract. Mutations of the ß-globin RNA activator abrogated PKR activation and profoundly affected mRNA splicing efficiency. PKR depletion abrogated splicing and spliceosome assembly; recombinant PKR effectively restored splicing. Excision of the first intron of ß-globin induces strand displacement within the RNA activator of PKR by a sequence from exon 2, a structural rearrangement that silences the ability of spliced ß-globin mRNA to activate PKR. Thus, the ability to activate PKR is transient, serving solely to enable splicing. α-Globin pre-mRNA splicing is controlled likewise but positions of PKR activator and silencer are reversed, demonstrating evolutionary flexibility in how PKR activation regulates globin mRNA splicing through eIF2α phosphorylation.

mRNA encoding Sec61ß, a tail-anchored protein, is localized on the endoplasmic reticulum.

Although one pathway for the post-translational targeting of tail-anchored proteins to the endoplasmic reticulum (ER) has been well defined, it is unclear whether additional pathways exist. Here, we provide evidence that a subset of mRNAs encoding tail-anchored proteins, including Sec61ß and nesprin-2, is partially localized to the surface of the ER in mammalian cells. In particular, Sec61b mRNA can be targeted to, and later maintained on, the ER using both translation-dependent and -independent mechanisms. Our data suggests that this process is independent of p180 (also known as RRBP1), a known mRNA receptor on the ER, and the transmembrane domain recognition complex (TRC) pathway components, TRC40 (also known as ASNA1) and BAT3 (also known as BAG6). In addition, our data indicates that Sec61b mRNA might access translocon-bound ribosomes. Our results show that certain tail-anchored proteins are likely to be synthesized directly on the ER, and this facilitates their membrane insertion. Thus, it is clear that mammalian cells utilize multiple mechanisms to ensure efficient targeting of tail-anchored proteins to the surface of the ER.

Vav1 promotes lung cancer growth by instigating tumor-microenvironment cross-talk via growth factor secretion.

Vav1 is a signal transducer that functions as a scaffold protein and a regulator of cytoskeleton organization in the hematopoietic system, where it is exclusively expressed. Recently, Vav1 was shown to be involved in diverse human cancers, including lung cancer. We demonstrate that lung cancer cells that abnormally express Vav1 secrete growth factors in a Vav1-dependent manner. Transcriptome analysis demonstrated that Vav1 depletion results in a marked reduction in the expression of colony-stimulating-factor-1 (CSF1), a hematopoietic growth factor. The association between Vav1 expression and CSF1 was further supported by signal transduction experiments, supporting involvement of Vav1 in regulating lung cancer secretome. Blocking of ERK phosphorylation, led to a decrease in CSF1 transcription, thus suggesting a role for ERK, a downstream effector of Vav1, in CSF1 expression. CSF1-silenced cells exhibited reduced focus formation, proliferation abilities, and growth in NOD/SCID mice. CSF1-silenced H358 cells resulted in significantly smaller tumors, showing increased fibrosis and a decrease in tumor infiltrating macrophages. Finally, immunohistochemical analysis of primary human lung tumors revealed a positive correlation between Vav1 and CSF1 expression, which was associated with tumor grade. Additional results presented herein suggest a potential cross-talk between cancer cells and the microenvironment controlled by CSF1/Vav1 signaling pathways.

Vav1 fine tunes p53 control of apoptosis versus proliferation in breast cancer.

Vav1 functions as a signal transducer protein in the hematopoietic system, where it is exclusively expressed. Vav1 was recently implicated in several human cancers, including lung, pancreatic and neuroblasoma. In this study, we analyzed the expression and function of Vav1 in human breast tumors and breast cancer cell lines. Immunohistochemical analysis of primary human breast carcinomas indicated that Vav1 is expressed in 62% of 65 tumors tested and is correlated positively with estrogen receptor expression. Based on published gene profiling of 50 breast cancer cell lines, several Vav1-expressing cell lines were identified. RT-PCR confirmed Vav1 mRNA expression in several of these cell lines, yet no detectable levels of Vav1 protein were observed due to cbl-c proteasomal degradation. We used two of these lines, MCF-7 (Vav1 mRNA negative) and AU565 (Vav1 mRNA positive), to explore the effect of Vav1 expression on breast cell phenotype and function. Vav1 expression had opposite effects on function in these two lines: it reduced proliferation and enhanced cell death in MCF-7 cells but enhanced proliferation in AU565 cells. Consistent with these findings, transcriptome analysis revealed an increase in expression of proliferation-related genes in Vav1-expressing AU565 cells compared to controls, and an increase in apoptosis-related genes in Vav1-expressing MCF-7 cells compared with controls. TUNEL and γ-H2AX foci assays confirmed that expression of Vav1 increased apoptosis in MCF-7 cells but not AU565 cells and shRNA experiments revealed that p53 is required for this pro-apoptotic effect of Vav1 in these cells. These results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer and the p53 dependence of its pro-apoptotic effect in breast cells.

Human Vav1 expression in hematopoietic and cancer cell lines is regulated by c-Myb and by CpG methylation.

Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA). Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5' regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well.