Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation.

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.

Development of automated image analysis tools for verification of radiotherapy field accuracy with an electronic portal imaging device

The successful management of cancer with radiation relies on the accurate deposition of a prescribed dose to a prescribed anatomical volume within the patient. Treatment set-up errors are inevitable because the alignment of field shaping devices with the patient must be repeated daily up to eighty times during the course of a fractionated radiotherapy treatment. With the invention of electronic portal imaging devices (EPIDs), patient's portal images can be visualized daily in real-time after only a small fraction of the radiation dose has been delivered to each treatment field. However, the accuracy of human visual evaluation of low-contrast portal images has been found to be inadequate. The goal of this research is to develop automated image analysis tools to detect both treatment field shape errors and patient anatomy placement errors with an EPID. A moments method has been developed to align treatment field images to compensate for lack of repositioning precision of the image detector. A figure of merit has also been established to verify the shape and rotation of the treatment fields. Following proper alignment of treatment field boundaries, a cross-correlation method has been developed to detect shifts of the patient's anatomy relative to the treatment field boundary. Phantom studies showed that the moments method aligned the radiation fields to within 0.5mm of translation and 0.5$\sp\circ$ of rotation and that the cross-correlation method aligned anatomical structures inside the radiation field to within 1 mm of translation and 1$\sp\circ$ of rotation. A new procedure of generating and using digitally reconstructed radiographs (DRRs) at megavoltage energies as reference images was also investigated. The procedure allowed a direct comparison between a designed treatment portal and the actual patient setup positions detected by an EPID. Phantom studies confirmed the feasibility of the methodology. Both the moments method and the cross-correlation technique were implemented within an experimental radiotherapy picture archival and communication system (RT-PACS) and were used clinically to evaluate the setup variability of two groups of cancer patients treated with and without an alpha-cradle immobilization aid. The tools developed in this project have proven to be very effective and have played an important role in detecting patient alignment errors and field-shape errors in treatment fields formed by a multileaf collimator (MLC). ^

STRUCTURAL ANALYSIS OF THE TRANSCRIPTION PRODUCTS OF A TEMPERATURE-SENSITIVE PHENOTYPIC MUTANT OF MOLONEY SARCOMA VIRUS--MUSV TS110

Cells infected with a temperature sensitive phenotypic mutant of Moloney sarcoma virus (MuSVts110) exhibit a transformed phenotype at 33('(DEGREES)) and synthesize two virus specific proteins, p85('gag-mos), a gag-mos fusion protein and p58('gag), a truncated gag precursor protein (the gag gene codes for viral structural proteins and mos is the MuSV transforming gene). At 39('(DEGREES)) only p58('gag) is synthesized and the morphology of the cells is similar to uninfected NRK parental cells. Two MuSVts110 specific RNAs are made in MuSVts110-infected cells, one of 4.0 kb in length, the other of 3.5 kb. Previous work indicated that each of these RNAs arose by a single central deletion of parental MuSV genetic material, and that p58('gag) was made by the 4.0 kb RNA and p85('gag-mos) from the 3.5 kb RNA. The objective of my dissertation research was to map precisely the deletion boundaries of both of the MuSVts110 RNAs, and to determine the proper reading frame across both deletion borders. This work succeeded in arriving at the following conclusions: (a) Using S-1 nuclease analysis and primer extension sequencing, it was found that the 4.0 kb MuSVts110 RNA arose by a 1488 base deletion of 5.2 kb parental MuSV genomic RNA. This deletion resulted in an out of frame fusion of the gag and mos genes that resulted in the formation of a "stop" codon which causes termination of translation just beyond the c-terminus of the gag region. Thus, this RNA can only be translated into the truncated gag protein p58('gag). (b) S-1 analysis of RNA from cells cultivated at different temperatures demonstrated that the 4.0 kb RNA was synthesized at all temperatures but that synthesis of the 3.5 kb RNA was temperature sensitive. These observations supported the data derived from blot hybridization experiments the interpretation of which argued for the existence of a single provirus in MuSVts110 infected cells, and hence only a single primary transcript (the 4.0 kb RNA). (c) Analyses similar to those described in (a) above showed that the 3.5 kb RNA was derived from the 4.0 kb MuSVts110 RNA by a further deletion of 431 bases, fusing the gag and mos genes into a continuous reading frame capable of directing synthesis of the p85('gag-mos) protein. These sequence data and the presence of only one MuSVts110-specific provirus, indicate that a splice mechanism is employed to generate the 3.5 kb RNA since the gag and mos genes are observed to be fused in frame in this RNA. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

RNA binding characteristics and overall topology of the vaccinia virus polyadenylation complex

mRNA 3′ polyadenylation is central to mRNA biogenesis in prokaryotes and eukaryotes, and is implicated in numerous aspects of mRNA metabolism, including efficiency of mRNA export from the nucleus, message stability, and initiation of translation. However, due to the great complexity of the eukaryotic polyadenylation apparatus, the mechanisms of RNA 3 ′ end processing have remained elusive. Although the RNA processing reactions leading to polyadenylated messenger RNA have been studied in many systems, and much progress has been made, a complete understanding of the biochemistry of the poly(A) polymerase enzyme is still lacking. My research uses Vaccinia virus as a model system to gain a better understanding of this complicated polyadenylation process, which consist of RNA binding, catalysis and polymerase translocation. ^ Vaccinia virus replicates in the cytoplasm of its host cell, so it must employ its own poly(A) polymerase (PAP), a heterodimer of two virus encoded proteins, VP55 and VP39. VP55 is the catalytic subunit, adding 30 adenylates to a non-polyadenylated RNA in a rapid processive manner before abruptly changing to a slow, non-processive mode of adenylate addition and dissociating from the RNA. VP39 is the stimulatory subunit. It has no polyadenylation catalytic activity by itself, but when associated with VP55 it facilitates the semi-processive synthesis of tails several hundred adenylates in length. ^ Oligonucleotide selection and competition studies have shown that the heterodimer binds a minimal motif of (rU)2 (N)25 U, the “heterodimer binding motif”, within an oligonucleotide, and its primer selection for polyadenylation is base-type specific. ^ Crosslinking studies using photosensitive uridylate analogs show that within a VP55-VP39-primer ternary complex, VP55 comes into contact with all three required uridylates, while VP39 only contacts the downstream uridylate. Further studies, using a backbone-anchored photosensitive crosslinker show that both PAP subunits are in close proximity to the downstream −10 to −21 region of 50mer model primers containing the heterodimer binding motif. This equal crosslinking to both subunits suggests that the dimerization of VP55 and VP39 creates either a cleft or a channel between the two subunits through which this region of RNA passes. ^ Peptide mapping studies of VP39 covalently crosslinked to the oligonucleotide have identified residue R107 as the amino acid in close proximity to the −10 uridylate. This helps us project a conceptual model onto the known physical surface of this subunit. In the absence of any tertiary structural data for VP55, we have used a series of oligonucleotide selection assays, as well as crosslinking, nucleotide transfer assays, and gel shift assays to gain insight into the requirements for binding, polyadenylation and translocation. Collectively, these data allow us to put together a comprehensive model of the structure and function of the polyadenylation ternary complex consisting of VP39, VP55 and RNA. ^

Identification and characterization of distinct classes of cAMP-pulse-induced genes in Dictyostelium discoideum development

The unicellular amoeba Dictyostelium discoideum embarks on a developmental program upon starvation. During development, extracellular oscillatory cAMP signaling orchestrates the chemotaxis-mediated aggregation of ∼105 amoebae and is required for optimal induction of so-called pulse-induced genes. This requirement for pulsatile CAMP reflects adaptation of the cAMP-receptor-mediated pathways that regulate these genes. Through examination of a collection of pulse-induced genes, we defined two distinct gene classes based on their induction kinetics and the impact of mutations that impair PKA signaling. The first class (represented by D2 and prtA) is highly dependent on PKA signaling, whereas the second class (represented by carA, gpaB, and acaA) is not. Analysis of expression kinetics revealed that these classes are sequentially expressed with the PKA-independent genes peaking in expression before the PKA-dependent class. Experiments with cycloheximide, an inhibitor of translation, demonstrated that the pulse induction of both classes depends on new protein synthesis early in development. carA and gpaB also exhibit pulse-independent, starvation-induced expression which, unlike their pulse induction, was found to be insensitive to cycloheximide added at the outset of starvation. This result indicates that the mechanism of starvation induction pre-exists in growing cells and is distinct from the pulse induction mechanism for these genes. In order to identify cis-acting elements that are critical for induction of carA, we constructed a GFP reporter controlled by a 914-base-pair portion of its promoter and verified that its expression was PKA-independent, pulse-inducible, and developmentally regulated like the endogenous carA gene. By a combination of truncation, internal deletion, and site-directed mutation, we defined several distinct functional elements within the carA promoter, including a 39-bp region required for pulse induction between base pairs -321 and -282 (relative to the transcription start site), a 131-bp region proximal to the start site that is sufficient for starvation induction, and two separate enhancer domains. Identification of factors that interact with these promoter elements and genetic approaches exploiting the GFP reporter described here should help complete our understanding of the mechanisms regulating these genes, including adaptation mechanisms that likely also govern chemotaxis of Dictyostelium and mammalian cells. ^

Mechanism of translationally coupled mRNA turnover mediated by c-fos protein-coding-region determinant

Proto-oncogene c-fos is a member of the class of early-response genes whose transient expression plays a crucial role in cell proliferation, differentiation, and apoptosis. Degradation of c- fos mRNA is an important mechanism for controlling c-fos expression. Rapid mRNA turnover mediated by the protein-coding-region determinant (mCRD) of the c-fos transcript illustrates a functional interplay between mRNA turnover and translation that coordinately influences the fate of cytoplasmic mRNA. It is suggested that mCRD communicates with the 3′ poly(A) tail via an mRNP complex comprising mCRD-associated proteins, which prevents deadenylation in the absence of translation. Ribosome transit as a result of translation is required to alter the conformation of the mRNP complex, thereby eliciting accelerated deadenylation and mRNA decay. To gain further insight into the mechanism of mCRD-mediated mRNA turnover, Unr was identified as an mCRD-binding protein, and its binding site within mCRD was characterized. Moreover, the functional role for Unr in mRNA decay was demonstrated. The result showed that elevation of Unr protein level in the cytoplasm led to inhibition of mRNA destabilization by mCRD. In addition, GST pull-down assay and immuno-precipitation analysis revealed that Unr interacted with PABP in an RNA-independent manner, which identified Unr as a novel PABP-interacting protein. Furthermore, the Unr interacting domain in PABP was characterized. In vivo mRNA decay experiments demonstrated a role for Unr-PABP interaction in mCRD-mediated mRNA decay. In conclusion, the findings of this study provide the first evidence that Unr plays a key role in mCRD-mediated mRNA decay. It is proposed that Unr is recruited by mCRD to initiate the formation of a dynamic mRNP complex for communicating with poly(A) tail through PABP. This unique mRNP complex may couple translation to mRNA decay, and perhaps to recruit the responsible nuclease for deadenylation. ^

The yeast STM1 protein binds G*G multiplex nucleic acids in vitro and associates with ribosomes in vivo

The formation of triple helical, or triplex DNA has been suggested to occur in several cellular processes such as transcription, replication, and recombination. Our laboratory previously found proteins in HeLa nuclear extracts and in S. cerevisiae whole cell extracts that avidly bound a Purine-motif (Pu) triplex probe in gel shift assays, or EMSA. In order to identify a triplex DNA-binding protein, we used conventional and affinity chromatography to purify the major Pu triplex-binding protein in yeast. Peptide microsequencing and data base searches identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a Pu triplex-binding protein was obtained by EMSA using both recombinant Stm1p and whole cell extracts from stm1Δ yeast. Stm1p had previously been identified as G4p2, a G-quartet DNA- and RNA-binding protein. To study the cellular role and identify the nucleic acid ligand of Stm1p in vivo, we introduced an HA epitope at either the N- or C-terminus of Stm1p and performed immunoprecipitations with the HA.11 mAb. Using peptide microsequencing and Northern analysis, we positively identified a subset of both large and small subunit ribosomal proteins and all four rRNAs as associating with Stm1p. DNase I treatment did not affect the association of Stm1p with ribosomal components, but RNase A treatment abolished the association with all ribosomal proteins and RNA, suggesting this association is RNA-dependent. Sucrose gradient fractionation followed by Western and EMSA analysis confirmed that Stm1p associates with intact 80S monosomes, but not polysomes. The presence of additional, unidentified RNA in the Stm1p-immunoprecipitate, and the absence of tRNAs and elongation factors suggests that Stm1p binds RNA and could be involved in the regulation of translation. Immunofluorescence microscopy data showed Stm1p to be located throughout the cytoplasm, with a specific movement to the bud during the G2 phase of the cell cycle. A dramatically flocculent, large cell phenotype is observed when Stm1p has a C-terminal HA tag in a protease-deficient strain background. When STM1 is deleted in this background, the same phenotype is not observed and the deletion yeast grow very slowly compared to the wild-type. These data suggest that STM1 is not essential, but plays a role in cell growth by interacting with an RNP complex that may contain G*G multiplex RNA. ^

The role of post-translation regulation of Twist expression in the tumor progression of squamous cell carcinoma of head and neck

Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://scansite.mit.edu/motifscan_seq.phtml ), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addition, mutation of the putative CK2 phosphorylation site (S18/S20A) in TWIST decreased the amount of phospho-ATP incorporated by TWIST in an in vitro kinase assay, and altered TWIST stability. In Boyd chamber migration assay and wound-healing assay, the CK2 inhibitor, DMAT, was found to decrease the motility of IL-6 stimulated SCCHN cells and over expression of either a wild-type or the hyperphosphorylated mimicking mutant S18/20D –Twist rather than the hypo-phosphorylated mimicking mutant S18/20A-Twist can promote SCCHN cell motility.To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway.

The role of specific regions of ribosomal RNAs in translation termination

The ribosome is a molecular machine that produces proteins in a cell. It consists of RNAs (rRNAs) and proteins. The rRNAs have been implicated in various aspects of protein biosynthesis supporting the idea that they function directly in translation. In this study the direct involvement of rRNA in translation termination was hypothesized and both genetic and biochemical strategies were designed to test this hypothesis. As a result, several regions of rRNAs from both ribosomal subunits were implicated in termination. More specifically, the mutation G1093A in an RNA of the large subunit (23S rRNA) and the mutation C1054A in the small subunit RNA (16S rRNA) of the Escherichia coli ribosome, were shown to affect the binding of the proteins that drive termination, RF1 and RF2. These mutations also caused defects in catalysis of peptidyl-tRNA hydrolysis, the last step of termination. Furthermore, the mutations affected the function of RF2 to a greater extent than that of RF1, a striking result considering the similarity of the RFs. The major defect in RF2 function was consistent with in vivo characteristics of the mutants and can be explained by the inability of the mutant rRNA sites to activate the hydrolytic center, that is the catalytic site for peptidyl-tRNA hydrolysis. Consistent with this explanation is the possibility of a direct interaction between the G1093-region (domain II of 23S rRNA) and the hydrolytic center (most likely domains IV–VI of 23S rRNA). To test that interaction hypothesis selections were performed for mutations in domains IV–VI that compensated for the growth defects caused by G1093A. Several compensatory mutations were isolated which not only restored growth in the presence of G1093A but also appeared to compensate for the termination defects caused by the G1093A. Therefore these results provided genetic evidence for an intramolecular interaction that might lead to peptidyl-tRNA hydrolysis. Finally, a new approach to the study of rRNA involvement in termination was designed. By screening a library of rRNA fragments, a fragment of the 23S rRNA (nt 74-136) was identified that caused readthrough of UGA. The antisense RNA fragment produced a similar effect. The data implicated the corresponding segment of intact 23S rRNA in termination. ^

THE TRANSLATION PRODUCTS OF MOLONEY MURINE SARCOMA VIRUS 124 GENOMIC RNA

Murine sarcoma viruses constitute a class of replication-defective retroviruses. Cellular transformation may be induced by these viruses in vitro; whereas, fibrosarcomas may result in animals infected with them in vivo (Tooze, 1973; Bishop, 1978). Hybridization studies suggest that murine sarcoma viruses arose by recombination between nondefective murine leukemia virus sequences and certain cellular sequences present in uninfected mouse cells (Hu et al., 1977). A specific gene product, however, has not been implicated in murine sarcoma virus transformation.^ One line of murine sarcoma virus-producing cells, Mo-MuSV-clone 124, (Ball et al., 1973), was studied biochemically because it mainly produces the sarcoma virus as a pseudotype packaged with helper murine leukemia virus proteins. The sarcoma viral RNA was translated in a sophisticated cell-free protein synthesizing system (Murphy and Arlinghaus, 1978). The translation products were analyzed by a number of techniques, including electrophoresis in denaturing gels of SDS polyacrylamide, immunoprecipitation, and peptide mapping. The major products of the total RNA purified from the virus preparation were shown to have molecular weights of about 63,000 (P63('gag)), 42,000 (P42), 40,000 (P40), 38,000 (P38), and 23,000 (P23). The size class of mRNA coding for each of the cell-free products was estimated using a poly(A) selection technique and sucrose gradient fractionation. These analyses were used to localize the coding information related to each of the in vitro synthesized cell-free products within the sarcoma virus genome.^ The major findings of these studies were: (1) the 5' half of the sarcoma viral RNA codes for the 63,000 dalton polypeptide and 42,000 - 38,000 dalton polypeptides derived from the "gag" gene; and (2) the 3' half of the sarcoma viral RNA codes for a 38,000 dalton polypeptide and possibly derived from the cellular acquired sequences. ^

The Spanish translation, cultural adaptation and validation of the Powe Fatalism Inventory

Background/significance. The scarcity of reliable and valid Spanish language instruments for health related research has hindered research with the Hispanic population. Research suggests that fatalistic attitudes are related to poor cancer screening behaviors and may be one reason for low participation of Mexican-Americans in cancer screening. This problem is of major concern because Mexican-Americans constitute the largest Hispanic subgroup in the U.S.^ Purpose. The purposes of this study were: (1) To translate the Powe Fatalism Inventory, (PFI) into Spanish, and culturally adapt the instrument to the Mexican-American culture as found along the U.S.-Mexico border and (2) To test the equivalence between the Spanish translated, culturally adapted version of the PFI and the English version of the PFI to include clarity, content validity, reading level and reliability.^ Design. Descriptive, cross-sectional.^ Methods. The Spanish language translation used a translation model which incorporates a cultural adaptation process. The SPFI was administered to 175 bilingual participants residing in a midsize, U.S-Mexico border city. Data analysis included estimation of Cronbach's alpha, factor analysis, paired samples t-test comparison and multiple regression analysis using SPSS software, as well as measurement of content validity and reading level of the SPFI. ^ Findings. A reliability estimate using Cronbach's alpha coefficient was 0.81 for the SPFI compared to 0.80 for the PFI in this study. Factor Analysis extracted four factors which explained 59% of the variance. Paired t-test comparison revealed no statistically significant differences between the SPFI and PFI total or individual item scores. Content Validity Index was determined to be 1.0. Reading Level was assessed to be less than a 6th grade reading level. The correlation coefficient between the SPFI and PFI was 0.95.^ Conclusions. This study provided strong psychometric evidence that the Spanish translated, culturally adapted SPFI is an equivalent tool to the English version of the PFI in measuring cancer fatalism. This indicates that the two forms of the instrument can be used interchangeably in a single study to accommodate reading and speaking abilities of respondents. ^

Translation and cultural adaptation of the Stigma-Devaluation scale for use among family caregivers of mentally ill relatives in Jordan

Background/significance. Mental illness stigma is a matter of great concern to family caregivers. Few research studies have been conducted in the Arab World on family caregivers' perception of stigma associated with caring for a mentally ill relative. Review of the literature on measurement of the concept of stigma related to caring for a mentally ill relative yielded no instrument appropriate for use in a Jordanian sample. Reliable and valid instruments to measure stigma perception among family caregivers are needed for research and practice, particularly in Arabic speaking populations. ^ Purpose. The purposes of this study were: (1) translate the Stigma-Devaluation scale (SDS) into Arabic, modifying it to accurately reflect the cultural parameters specific to Jordan, and (2) test the reliability, the content and construct validity of the Arabic version of the SDS for use among a sample of family members of mentally ill relatives in Jordan. ^ Design. Methodologic, cross-sectional. ^ Methods. The SDS was translated into Arabic language, modified and culturally adapted to the Jordanian culture by a translation model which incorporates a cultural adaptation process. The Arabic SDS was evaluated in a sample of 164 family caregivers in the outpatient mental health clinic in Irbid-Jordan. Cronbach's alpha estimation of internal consistency was used to assess the reliability of the SDS. Construct validity was determined by confirmatory factor analysis (CFA). Measurements of content validity and reading level of the Arabic SDS were included. ^ Findings. Content Validity Index was determined to be 1.0. Reading level of the Arabic SDS was considered at a 6th grade or lower Cronbach's alpha coefficient of the modified Arabic SDS total scale was .87. Initial results of CFA did not fully support the proposed factor structures of the SDS or its subscales. After modifications, the indices indicated that the modified model of each subscale had satisfactory fit. ^ Conclusion. This study provided psychometric evidence that the modified Arabic SDS translated and culturally adapted instrument, is valid and conceptually consistent with the content of the original English SDS in measuring stigma perception among families of mentally ill relatives in Jordan. ^