Role of the small subunit processome in the maintenance of pluripotent stem cells.

RNA-binding proteins (RBPs) play integral roles in gene regulation, yet only a small fraction of RBPs has been studied in the context of stem cells. Here we applied an RNAi screen for RBPs in mouse embryonic stem cells (ESCs) and identified 16 RBPs involved in pluripotency maintenance. Interestingly, six identified RBPs, including Krr1 and Ddx47, are part of a complex called small subunit processome (SSUP) that mediates 18S rRNA biogenesis. The SSUP components are preferentially expressed in stem cells and enhance the global translational rate, which is critical to sustain the protein levels of labile pluripotency factors such as Nanog and Esrrb. Furthermore, the SSUP proteins are required for efficient reprogramming of induced pluripotent stem cells. Our study uncovers the role of the SSUP and the importance of translational control in stem cell fate decision.

Identification of frequently mutated genes with relevance to nonsense mediated mRNA decay in the high microsatellite instability cancers.

Frameshift mutations at coding mononucleotide repeats (cMNR) are frequent in high-microsatellite instability (MSI-H) cancers. Frameshift mutations in cMNR result in the formation of a premature termination codon (PTC) in the transcribed mRNA, and these abnormal mRNAs are generally degraded by nonsense mediated mRNA decay (NMD). We have identified novel genes that are frequently mutated at their cMNR by blocking NMD in two MSI-H cancer cell lines. After blocking NMD, we screened for differentially expressed genes using DNA microarrays, and then used database analysis to select 28 candidate genes containing cMNR with more than 9 nucleotide repeats. cMNR mutations have not been previously reported in MSI-H cancers for 15 of the 28 genes. We analyzed the cMNR mutation of each of the 15 genes in 10 MSI-H cell lines and 21 MSI-H cancers, and found frequent mutations of 12 genes in MSI-H cell lines and cancers, but not in microsatellite stable (MSS) cancers. Among these genes, the most frequently mutated in MSI-H cell lines were MLL3 (70%), PHACTR4 (70%), RUFY2 (50%) and TBC1D23 (50%). MLL3, which has already been implicated in cancer, had the highest mutation frequency in MSI-H cancers (48%). Our combined approach of NMD block, database search, and mutation analysis has identified a large number of genes mutated in their cMNR in MSI-H cancers. The identified mutations are expected to contribute to MSI-H tumorigenesis by causing an absence of gene expression or low gene dosage effects.

Tudor domain-containing protein 4 as a potential cancer/testis antigen in liver cancer.

The poor prognosis of liver cancer demands the development of new diagnostic markers and therapeutic strategies. Cancer/testis (CT) antigens are expressed in the testis and cancerous tissues, but not in adult somatic cells. Given their tumor-specific expression, CT antigens are potential molecular markers for tumor diagnosis and targets for cancer immunotherapy. To identify novel CT antigens for liver cancer, we examined mRNA expression of hitherto unknown CT antigen candidates, tudor domain-containing protein (TDRD) 1, 4 and 5 in three types of liver cancer; hepatocellular carcinoma (HCC, n = 28), cholangiocarcinoma (CC, n = 5) and combined HCC-CC (n = 8), with matched non-tumorous liver tissues. The TDRD1, 4 and 5 are known as being specifically expressed in the testis. TDRD1 and 5 are essential for male germ cell development. On RT-PCR analysis, TDRD1 mRNA was expressed in both HCCs and non-tumorous liver tissues, and TDRD5 mRNA was expressed in normal colonic and gastric mucosal tissues. Thus, TDRD1 and TDRD5 are not candidates for CT antigens. TDRD4 mRNA was expressed in the testis but not in other normal tissues, including colonic mucosa, gastric mucosa, and liver tissues. TDRD4 mRNA was expressed in 7 of the 41 liver cancers: 4 HCCs, 1 CC and 2 combined HCC-CCs. The TDRD4 mRNA expression was not significantly associated with patient age, tumor size, pathologic stages, hepatitis B virus infection, or CD133 expression. In conclusion, TDRD4 mRNA is expressed in a subset of liver cancers, and TDRD4 is a candidate CT antigen for liver cancer.

MicroRNA expression profile of gastrointestinal stromal tumors is distinguished by 14q loss and anatomic site.

MicroRNAs are known to regulate gene expression. Although unique microRNA expression profiles have been reported in several tumors, little is known about microRNA expression profiles in GISTs. To evaluate the relationship between microRNA expression and clinicopathologic findings of GISTs, we analyzed the microRNA expression profiles of GISTs. We used fresh frozen tissues from 20 GISTs and analyzed KIT and PDGFRA mutations and chromosomal loss status. MicroRNA expression was analyzed using a microRNA chip containing 470 microRNAs. Using unsupervised hierarchical clustering analysis, we found four distinct microRNA expression patterns in our 20 GISTs. Six GISTs that did not have 14q loss formed a separate cluster. In the 14 GISTs with 14q loss, 5 small bowel GISTs formed a separate cluster and the remaining 9 GISTs could be divided into two groups according to frequent chromosomal losses and tumor risk. We found 73 microRNAs that were significantly down-regulated in the GISTs with 14q loss; 38 of these microRNAs are encoded on 14q. We also found many microRNAs that were down-regulated in small bowel and high-risk group GISTs. Most of the microRNAs down-regulated in the high-risk group and small bowel GISTs are known to be involved in tumor progression, specifically by stimulating mitogen-activated protein kinase (MAPK) and the cell cycle. The microRNA expression patterns of GISTs are closely related to the status of 14q loss, anatomic site, and tumor risk. These findings suggest that microRNA expression patterns can differentiate several subsets of GISTs.

Selective translational repression of truncated proteins from frameshift mutation-derived mRNAs in tumors.

Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs). Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD) system. However, PTCs located within 50-55 nucleotides of the last exon-exon junction are not recognized by NMD (NMD-irrelevant), and some PTC-containing mRNAs can escape from the NMD system (NMD-escape). We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.

Stress-activated miR-204 governs senescent phenotypes of chondrocytes to promote osteoarthritis development.

A progressive loss of cartilage matrix leads to the development of osteoarthritis (OA). Matrix homeostasis is disturbed in OA cartilage as the result of reduced production of cartilage-specific matrix and increased secretion of catabolic mediators by chondrocytes. Chondrocyte senescence is a crucial cellular event contributing to such imbalance in matrix metabolism during OA development. Here, we identify miR-204 as a markedly up-regulated microRNA in OA cartilage. miR-204 is induced by transcription factors GATA4 and NF-κB in response to senescence signals. Up-regulated miR-204 simultaneously targets multiple components of the sulfated proteoglycan (PG) biosynthesis pathway, effectively shutting down PG anabolism. Ectopic expression of miR-204 in joints triggers spontaneous cartilage loss and OA development, whereas miR-204 inhibition ameliorates experimental OA, with concomitant recovery of PG synthesis and suppression of inflammatory senescence-associated secretory phenotype (SASP) factors in cartilage. Collectively, we unravel a stress-activated senescence pathway that underlies disrupted matrix homeostasis in OA cartilage.

Differential gene expression profiles of metastases in paired primary and metastatic colorectal carcinomas.

BACKGROUND AND METHODS: Despite the overwhelming clinical significance of metastases, the cellular and molecular mechanisms involved are largely unknown. In order to define significant differences between primary colon carcinomas and their metastases, we analyzed gene expression profiles of 12 sets of triple-paired tissues using 19 K human oligonucleotide microarrays. A total of 36 microarray experiments were analyzed by unsupervised two-way hierarchical clustering and multi-dimensional scaling (MDS). RESULTS: Both methods completely distinguished normal mucosa from carcinoma, but failed to demonstrate a complete classification of primary and metastatic carcinomas. We found a separable tendency to be classified into the primary and metastatic colon carcinomas by MDS. In supervised hierarchical clustering, we identified 80 genes that were differentially expressed between paired primary and metastatic colon carcinomas. The 80 identified genes also successfully distinguished three validation sets of primary and lung-metastatic colon carcinomas. A specific set of genes was identified that distinguished the metastasis from the corresponding primary tumor in nearly half of the metastases analyzed. CONCLUSIONS: We suggest that a more accurate model of the metastatic potential is based on a global tumor expression pattern along with the appearance of distinct metastatic variants. This molecular profiling may be useful for the future study of colon cancer metastasis.

mRNAs containing NMD-competent premature termination codons are stabilized and translated under UPF1 depletion.

mRNAs containing premature termination codons (PTCs) are rapidly degraded through nonsense-mediated mRNA decay (NMD). However, some PTC-containing mRNAs evade NMD, and might generate mutant proteins responsible for various diseases, including cancers. Using PTC-containing human genomic ß-globin constructs, we show that a fraction (~30%) of PTC-containing mRNAs expressed from NMD-competent PTC-containing constructs were as stable as their PTC-free counterparts in a steady state. These PTC-containing mRNAs were monosome-enriched and rarely contributed to expression of mutant proteins. Expression of trace amounts of mutant proteins from NMD-competent PTC-containing constructs was not affected by inhibition of eIF4E-dependent translation and such expression was dependent on a continuous influx of newly synthesized PTC-containing mRNAs, indicating that truncated mutant proteins originated primarily in the pioneer round of translation. The generation of mutant proteins was promoted by UPF1 depletion, which induced polysome association of PTC-containing mRNAs, increased eIF4E-bound PTC-containing mRNA levels, and subsequent eIF4E-dependent translation. Our findings suggest that PTC-containing mRNAs are potent and regulatable sources of mutant protein generation.

Identification and selective degradation of neopeptide-containing truncated mutant proteins in the tumors with high microsatellite instability.

PURPOSE: Frameshift mutations in coding mononucleotide repeats (cMNR) are common in tumors with high microsatellite instability (MSI-H). These mutations generate mRNAs containing abnormal coding sequences and premature termination codons (PTC). Normally, mRNAs containing PTCs are degraded by nonsense-mediated mRNA decay (NMD). However, mRNAs containing PTCs located in the last exon are not subject to degradation by NMD (NMD-irrelevant). This study aimed to discover whether genes with frameshift mutations in the last exon generate truncated mutant proteins. EXPERIMENTAL DESIGN: We identified 66 genes containing cMNRs in the last exon by bioinformatic analysis. We found frequent insertion/deletion mutations in the cMNRs of 29 genes in 10 MSI-H cancer cell lines and in the cMNRs of 3 genes in 19 MSI-H cancer tissues. We selected 7 genes (TTK, TCF7L2, MARCKS, ASTE1, INO80E, CYHR1, and EBPL) for mutant mRNA expression analysis and 3 genes (TTK, TCF7L2, and MARCKS) for mutant protein expression analysis. RESULTS: The PTC-containing NMD-irrelevant mRNAs from mutated genes were not degraded. However, only faint amounts of endogenous mutant TTK and TCF7L2 were detected, and we failed to detect endogenous mutant MARCKS. By polysome analysis, we showed that mRNAs from genomic mutant MARCKS constructs are normally translated. After inhibiting 3 protein degradation pathways, we found that only inhibition of the proteasomal pathway facilitated the rescue of endogenous mutant TTK, TCF7L2, and MARCKS. CONCLUSIONS: Our findings indicate that cancer cells scavenge potentially harmful neopeptide-containing mutant proteins derived from NMD-irrelevant abnormal mRNAs via the ubiquitin-proteasome system, and these mutant proteins may be important substrates for tumor-specific antigens.

The RNA-binding protein repertoire of embryonic stem cells.

RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and yet annotation of RBPs is limited mainly to those with known RNA-binding domains. To systematically identify the RBPs of embryonic stem cells (ESCs), we here employ interactome capture, which combines UV cross-linking of RBP to RNA in living cells, oligo(dT) capture and MS. From mouse ESCs (mESCs), we have defined 555 proteins constituting the mESC mRNA interactome, including 283 proteins not previously annotated as RBPs. Of these, 68 new RBP candidates are highly expressed in ESCs compared to differentiated cells, implicating a role in stem-cell physiology. Two well-known E3 ubiquitin ligases, Trim25 (also called Efp) and Trim71 (also called Lin41), are validated as RBPs, revealing a potential link between RNA biology and protein-modification pathways. Our study confirms and expands the atlas of RBPs, providing a useful resource for the study of the RNA-RBP network in stem cells.

Differentially expressed proteins in gastrointestinal stromal tumors with KIT and PDGFRA mutations.

Most gastrointestinal stromal tumors (GIST) have activating mutations in either KIT or PDGFRA. However, a small subset of GIST lacks either mutation. To investigate the molecular characteristics of GIST according to mutation type, protein expression profiles in 12 GIST (2 cases with PDGFRA mutations, 8 cases with KIT mutations and 2 cases lacking either mutation) were analyzed using 2-DE and MALDI-TOF-MS. Comparative analysis of the respective spot patterns using 2-DE showed that 15 proteins were differently expressed according to the mutation status. Expression levels of septin and heat shock protein (HSP) 27 were increased in GIST with KIT mutations and annexin V was overexpressed in GIST lacking either mutation. Among the 15 proteins, overexpression of 5 proteins [annexin V, high mobility group protein 1 (HMGB1), C13orf2, glutamate dehydrogenase 1 and fibrinogen beta chain] and decreased expression of RoXaN correlated with a higher tumor grade. These findings suggest that differential protein expression can be used as a diagnostic biomarker. Moreover, it may play a role in the development and progression of GIST according to activating mutation type, as these proteins have been shown to be involved in tumor metastasis, apoptosis and immune response.

Suppression of human selenium-binding protein 1 is a late event in colorectal carcinogenesis and is associated with poor survival.

The purpose of this study was to analyze altered protein expression in cancer tissues and determine its relationship to prognosis in colorectal carcinomas. We performed proteomic expression analysis on 14 colorectal carcinomas and matched nontumorous colonic mucosa by 2-DE and MALDI-TOF-MS. Comparative analysis of the respective spot patterns on 2-DE showed 14 spots that were markedly changed in the colorectal carcinomas. Among them, selenium-binding protein 1 (SELENBP1) was markedly decreased in 12 (85%) carcinomas. The reduced expression of SELENBP1 was further supported by Western blot analysis and immunohistochemistry. Suppression of SELENBP1 was further analyzed in another eight-paired adenomas and carcinomas from the same patients using Western blot analysis and immunohistochemistry, and revealed that one adenoma and seven carcinomas exhibited markedly reduced SELENBP1 expression. Patients with low levels of SELENBP1 expression had significantly lower overall survival rates (72 vs. 85%, p = 0.021) among the 240 stages II and III colorectal carcinomas by using tissue microarray analysis. Our findings indicate that suppression of SELENBP1 is a frequent and late event in colorectal carcinogenesis, and may contribute to the rapid progression of colorectal carcinoma.