LRPPRC/SLIRP suppresses PNPase-mediated mRNA decay and promotes polyadenylation in human mitochondria.

In human mitochondria, 10 mRNAs species are generated from a long polycistronic precursor that is transcribed from the heavy chain of mitochondrial DNA, in theory yielding equal copy numbers of mRNA molecules. However, the steady-state levels of these mRNAs differ substantially. Through absolute quantification of mRNAs in HeLa cells, we show that the copy numbers of all mitochondrial mRNA species range from 6000 to 51,000 molecules per cell, indicating that mitochondria actively regulate mRNA metabolism. In addition, the copy numbers of mitochondrial mRNAs correlated with their cellular half-life. Previously, mRNAs with longer half-lives were shown to be stabilized by the LRPPRC/SLIRP complex, which we find that cotranscriptionally binds to coding sequences of mRNAs. We observed that the LRPPRC/SLIRP complex suppressed 3' exonucleolytic mRNA degradation mediated by PNPase and SUV3. Moreover, LRPPRC promoted the polyadenylation of mRNAs mediated by mitochondrial poly(A) polymerase (MTPAP) in vitro. These findings provide a framework for understanding the molecular mechanism of mRNA metabolism in human mitochondria.

Identification and functional analyses of 11,769 full-length human cDNAs focused on alternative splicing.

We analyzed diversity of mRNA produced as a result of alternative splicing in order to evaluate gene function. First, we predicted the number of human genes transcribed into protein-coding mRNAs by using the sequence information of full-length cDNAs and 5'-ESTs and obtained 23 241 of such human genes. Next, using these genes, we analyzed the mRNA diversity and consequently sequenced and identified 11 769 human full-length cDNAs whose predicted open reading frames were different from other known full-length cDNAs. Especially, 30% of the cDNAs we identified contained variation in the transcription start site (TSS). Our analysis, which particularly focused on multiple variable first exons (FEVs) formed due to the alternative utilization of TSSs, led to the identification of 261 FEVs expressed in the tissue-specific manner. Quantification of the expression profiles of 13 genes by real-time PCR analysis further confirmed the tissue-specific expression of FEVs, e.g. OXR1 had specific TSS in brain and tumor tissues, and so on. Finally, based on the results of our mRNA diversity analysis, we have created the FLJ Human cDNA Database. From our result, it has been understood mechanisms that one gene produces suitable protein-coding transcripts responding to the situation and the environment.

Novel in vitro protein fragment complementation assay applicable to high-throughput screening in a 1536-well format.

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/beta-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC(50) value of 0.020 microM and did not inhibit PPI between TCF7/beta-catenin and PAC1/PAC2 even at a concentration of 250 microM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library.

Novel clusters of highly expressed genes accompany genomic amplification in breast cancers.

Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies.

Differential responses of normal human coronary artery endothelial cells against multiple cytokines comparatively assessed by gene expression profiles.

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1beta, tumor necrosis factor-alpha, interferon-beta, interferon-gamma, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions.

Comprehensive analysis of the ICEN (Interphase Centromere Complex) components enriched in the CENP-A chromatin of human cells.

The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.

Identification of a

The introduction of a human chromosome 1 via microcell-mediated chromosome transfer (MMCT) induces the cellular senescence in mouse melanoma B16-F10 cells. The senescent cells maintained still the telomerase activity, which is frequently associated with immortal growth of human cells, suggesting that a telomerase-independent mechanism is involved in the senescence observed in this mouse cell line. To map the senescence-inducing gene to a specific chromosomal region, we took two experimental approaches: identification of a minimal region with the senescence-inducing activity via MMCT of a series of subchromosomal transferrable fragments (STFs), each consisting of a different profile of human chromosome 1-derived regions, and identification of a region commonly deleted from the transferred chromosome 1 in the revertant clones that escaped cellular senescence. These approaches identified a 2.7-3.0 Mb of senescence-inducing region shared among the active STFs and a 2.4-3.0 Mb of commonly deleted region in the revertant clones. These two regions overlapped each other to map the responsible gene at the 450 to 600-kb interval between UniSTS93710 and D1S3542 on chromosome 1q42.3. This study provides essential information and materials for cloning and characterization of a novel senescence-inducing gene that functions in a telomerase-independent pathway, which is likely to be conserved between mice and humans.