Finn Cilius Nielsen

A Family of Insulin-Like Growth Factor II mRNA-Binding Proteins Represses Translation in Late Development

ABSTRACT Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5′ untranslated regions (5′ UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5′ UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5′ UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growth-dependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 –luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5′ UTR-binding proteins control IGF-II biosynthesis during late mammalian development.

p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC

Aberrant oncogene activation induces cellular senescence, an irreversible growth arrest that acts as a barrier against tumorigenesis. To identify microRNAs (miRNAs) involved in oncogene-induced senescence, we examined the expression of miRNAs in primary human TIG3 fibroblasts after constitutive activation of B-RAF. Among the regulated miRNAs, both miR-34a and miR-146a were strongly induced during senescence. Although members of the miR-34 family are known to be transcriptionally regulated by p53, we find that miR-34a is regulated independently of p53 during oncogene-induced senescence. Instead, upregulation of miR-34a is mediated by the ETS family transcription factor, ELK1. During senescence, miR-34a targets the important proto-oncogene MYC and our data suggest that miR-34a thereby coordinately controls a set of cell cycle regulators. Hence, in addition to its integration in the p53 pathway, we show that alternative cancer-related pathways regulate miR-34a, emphasising its significance as a tumour suppressor.

RNA‐binding IMPs promote cell adhesion and invadopodia formation

Oncofetal RNA‐binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss‐of‐function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post‐transcriptional control of the transcripts. In particular, we show that a 5.0 kb CD44 mRNA contained multiple IMP‐binding sites in its 3′UTR, and following IMP depletion this species became unstable. Direct knockdown of the CD44 transcript mimicked the effect of IMPs on invadopodia, and we infer that CD44 mRNA stabilization may be involved in IMP‐mediated invadopodia formation. Taken together, our results indicate that RNA‐binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation.

Ascertainment Biases in SNP Chips Affect Measures of Population Divergence

Chip-based high-throughput genotyping has facilitated genome-wide studies of genetic diversity. Many studies have utilized these large data sets to make inferences about the demographic history of human populations using measures of genetic differentiation such as F(ST) or principal component analyses. However, the single nucleotide polymorphism (SNP) chip data suffer from ascertainment biases caused by the SNP discovery process in which a small number of individuals from selected populations are used as discovery panels. In this study, we investigate the effect of the ascertainment bias on inferences regarding genetic differentiation among populations in one of the common genome-wide genotyping platforms. We generate SNP genotyping data for individuals that previously have been subject to partial genome-wide Sanger sequencing and compare inferences based on genotyping data to inferences based on direct sequencing. In addition, we also analyze publicly available genome-wide data. We demonstrate that the ascertainment biases will distort measures of human diversity and possibly change conclusions drawn from these measures in some times unexpected ways. We also show that details of the genotyping calling algorithms can have a surprisingly large effect on population genetic inferences. We not only present a correction of the spectrum for the widely used Affymetrix SNP chips but also show that such corrections are difficult to generalize among studies.

NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction of all hematopoietic stem and progenitor populations. In contrast, more differentiated cells were only mildly affected in Upf2-null mice, suggesting that NMD is mainly essential for proliferating cells. Furthermore, we show that UPF2 loss resulted in the accumulation of nonproductive rearrangement by-products from the Tcrb locus and that this, as opposed to the general loss of NMD, was particularly detrimental to developing T-cells. At the molecular level, gene expression analysis showed that Upf2 deletion led to a profound skewing toward up-regulated mRNAs, highly enriched in transcripts derived from processed pseudogenes, and that NMD impacts on regulated alternative splicing events. Collectively, our data demonstrate a unique requirement of NMD for organismal survival.

Dwarfism and Impaired Gut Development in Insulin-Like Growth Factor II mRNA-Binding Protein 1-Deficient Mice

ABSTRACT Insulin-like growth factor II mRNA-binding protein 1 (IMP1) belongs to a family of RNA-binding proteins implicated in mRNA localization, turnover, and translational control. Mouse IMP1 is expressed during early development, and an increase in expression occurs around embryonic day 12.5 (E12.5). To characterize the physiological role of IMP1, we generated IMP1-deficient mice carrying a gene trap insertion in the Imp1 gene. Imp1−/− mice were on average 40% smaller than wild-type and heterozygous sex-matched littermates. Growth retardation was apparent from E17.5 and remained permanent into adult life. Moreover, Imp1−/− mice exhibited high perinatal mortality, and only 50% were alive 3 days after birth. In contrast to most other organs, intestinal epithelial cells continue to express IMP1 postnatally, and Imp1−/− mice exhibited impaired development of the intestine, with small and misshapen villi and twisted colon crypts. Analysis of target mRNAs and global expression profiling at E12.5 indicated that Igf2 translation was downregulated, whereas the postnatal intestine showed reduced expression of transcripts encoding extracellular matrix components, such as galectin- 1, lumican, tenascin-C, procollagen transcripts, and the Hsp47 procollagen chaperone. Taken together, the results demonstrate that IMP1 is essential for normal growth and development. Moreover, IMP1 may facilitate intestinal morphogenesis via regulation of extracellular matrix formation.

Expression but incomplete maturation of progastrin in colorectal carcinomas.

BACKGROUND To evaluate the hypothesis that gastrin is a local growth factor in colonic carcinomas, the expression of gastrin messenger RNA (mRNA) and peptides were examined in five human colon carcinoma cell lines, 12 solid colon carcinomas, and normal colonic tissue. METHODS Northern analysis, reverse-transcription PCR, and a library of sequence-specific radioimmunoassays were the principal methods. RESULTS Cell lines, tumors, and normal tissue all expressed a gastrin mRNA of 0.7 kilobases, and all cell lines contained incompletely processed progastrin (range, 17-54 fmol/10(6) cells). Two cell lines secreted progastrin into the media (LoVo, 25 +/- 3 pmol/L; HCT116; 12 +/- 2 pmol/L). Normal colonic tissue and all the solid tumors also contained progastrin, the concentration being higher in tumors (range, 0.4-2 pmol/g) than in normal tissue (range, 0.1-0.2 pmol/g). Only one tumor contained carboxyamidated gastrins. CONCLUSIONS Normal and neoplastic colonic mucosa both express the gastrin gene, but the posttranslational phase of expression is attenuated. The incomplete processing and low level of expression suggest that autocrine gastrin secretion has only minor significance for normal adult and most neoplastic colonic tissue.

IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas

Neurogenin 3 (Ngn3) is key for endocrine cell specification in the embryonic pancreas and induction of a neuroendocrine cell differentiation program by misexpression in adult pancreatic duct cells. We identify the gene encoding IA1, a zinc‐finger transcription factor, as a direct target of Ngn3 and show that it forms a novel branch in the Ngn3‐dependent endocrinogenic transcription factor network. During embryonic development of the pancreas, IA1 and Ngn3 exhibit nearly identical spatio‐temporal expression patterns. However, embryos lacking Ngn3 fail to express IA1 in the pancreas. Upon ectopic expression in adult pancreatic duct cells Ngn3 binds to chromatin in the IA1 promoter region and activates transcription. Consistent with this direct effect, IA1 expression is normal in embryos mutant for NeuroD1, Arx, Pax4 and Pax6, regulators operating downstream of Ngn3. IA1 is an effector of Ngn3 function as inhibition of IA1 expression in embryonic pancreas decreases the formation of insulin‐ and glucagon‐positive cells by 40%, while its ectopic expression amplifies neuroendocrine cell differentiation by Ngn3 in adult duct cells. IA1 is therefore a novel Ngn3‐regulated factor required for normal differentiation of pancreatic endocrine cells.

Molecular Composition of IMP1 Ribonucleoprotein Granules

Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100–300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly(A)-binding proteins, and mRNAs. Moreover granules contained CBP80 and factors belonging to the exon junction complex and lacked eIF4E, eIF4G, and 60 S ribosomal subunits, indicating that embodied mRNAs are not translated. Granules embodied mRNAs corresponding to about 3% of the human embryonic kidney 293 mRNA transcriptome. Messenger RNAs encoding proteins participating in the secretory pathway and endoplasmic reticulum-associated quality control, as well as ubiquitin-dependent metabolism, were enriched in the granules, reinforcing the concept of RNP granules as post-transcriptional operons.

Collection of blood, saliva, and buccal cell samples in a pilot study on the danish nurse cohort : Comparison of the response rate and quality of genomic DNA

In this study, we compared the response rates of blood, saliva, and buccal cell samples in a pilot study on the Danish nurse cohort and examined the quantity and quality of the purified genomic DNA. Our data show that only 31% of the requested participants delivered a blood sample, whereas 72%, 80%, and 76% delivered a saliva sample, buccal cell sample via mouth swabs, or buccal cell sample on FTA card, respectively. Analysis of purified genomic DNA by NanoDrop and agarose gel electrophoresis revealed that blood and saliva samples resulted in DNA with the best quality, whereas the DNA quality from buccal cells was low. Genotype and PCR analysis showed that DNA from 100% of the blood samples and 72% to 84% of the saliva samples could be genotyped or amplified, whereas none of the DNA from FTA cards and only 23% of the DNA from mouth swabs could be amplified and none of the DNA from swabs and 94% of the DNA from FTA cards could be genotyped. Our study shows that the response rate of self-collection saliva samples and buccal cell samples were much higher than the response rate of blood samples in our group of Danish nurses. However, only the quality of genomic DNA from saliva samples was comparable with blood samples as accessed by purity, genotyping, and PCR amplification. We conclude that the use of saliva samples is a good alternative to blood samples to obtain genomic DNA of high quality and it will increase the response rate considerably in epidemiologic studies. (Cancer Epidemiol Biomarkers Prev 2007;16(10):2072–6)