We performed a large-scale cDNA analysis to explore the transcriptome of the budding yeast Saccharomyces cerevisiae. We sequenced two cDNA libraries, one from the cells exponentially growing in a minimal medium and the other from meiotic cells. Both libraries were generated by using a vector-capping method that allows the accurate mapping of transcription start sites (TSSs). Consequently, we identified 11,575 TSSs associated with 3,638 annotated genomic features, including 3,599 ORFs, to suggest that most yeast genes have two or more TSSs. In addition, we identified 45 previously undescribed introns, including those affecting current ORF annotations and those spliced alternatively. Furthermore, the analysis revealed 667 transcription units in the intergenic regions and transcripts derived from antisense strands of 367 known features. We also found that 348 ORFs carry TSSs in their 3-halves to generate sense transcripts starting from inside the ORFs. These results indicate that the budding yeast transcriptome is considerably more complex than previously thought, and it shares many recently revealed characteristics with the transcriptomes of mammals and other higher eukaryotes. Thus, the genome-wide active transcription that generates novel classes of transcripts appears to be an intrinsic feature of the eukaryotic cells. The budding yeast will serve as a versatile model for the studies on these aspects of transcriptome, and the full-length cDNA clones can function as an invaluable resource in such studies.alternative splicing ͉ antisense transcript ͉ noncoding RNA ͉ transcription start site

Background: An ideal format to describe transcriptome would be its composition measured on the scale of absolute numbers of individual mRNAs per cell. It would help not only to precisely grasp the structure of the transcriptome but also to accelerate data exchange and integration.

SUMMARYMembrane-type 1 matrix metalloproteinase (MT1-MMP) is known to be internalized from cell surface, however, the fate of internalized MT1-MMP is still unknown. Here we demonstrate that at least a part of internalized MT1-MMP is targeted for lysosomal proteolysis. -dependent enzymes that are essential for extracellular matrix (ECM) turnover in normal and pathological conditions. MMP anchored to plasma membrane are subgrouped into membrane-type MMP (MT-MMP) [1][2][3][4][5]. Of all the MT-MMPs, MT1-MMP is believed to be a crucial for the invasion of malignant tumors [1,5,6].MT1-MMP activity is regulated by several distinct mechanisms. To express on cell surface, proMT1-MMP requires for being processed by proprotein convertases [7]. The enzymatic activity on the cell surface can be inhibited by the tissue inhibitors of metalloproteinases (TIMP)-2, -3, and -4 but not by TIMP-1. After MMP-2 activation, a part of MT1-MMP is down-regulated through processing by activated MMP-2 and autocatalytically [8]. Resent studies have revealed that the cell-surface MT1-MMP is regulated by internalization [9,10]. However, it is largely unknown whether MT1-MMP is degraded at endosomes/lysosomes, or whether internalized MT1-MMP is recycled back to the plasma membrane.Tetraspanins of integral membrane proteins with four predicted transmembrane domains comprises a large group of ubiquitously expressed proteins that function in diverse contexts such as T-and B-cell activation, and cell migration and proliferation [11]. CD63 is a member of tetraspanins and a well-established component of the late endosomal and lysosomal membranes [12]. CD63 is highly expressed in primary melanoma but weaker or absent in metastatic melanomas [13,14]. Ectopic expression of CD63 in melanoma cells suppresses their metastatic 4 potential in animal model system [15] . In the present study, we analyzed the fate of internalized MT1-MMP and the effect of CD63 on the stability of MT1-MMP and demonstrated that CD63 plays at least in part a role in promoting lysosomal proteolysis of MT1-MMP. MATERIALS AND METHODS Cell Immunoprecipitations and Immunoblottings.The cells were washed with ice-cold phosphate buffered saline (PBS), homogenized in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM Na 3 VO 4 , 1 mM NaF, 1% TritonX-100. The cell lysates were used for immunoprecipitation with anti-FLAG-M2 antibody. The immunoprecipitates were subjected to immunoblotting using indicated antibodies.Gelatin Zymography. Gelatin zymography was performed with SDS-polyacrylamide gel containing 0.1% gelatin as described previously [3]. Gelatinolytic activity was visualized as a negative staining with Coomassie Brilliant Blue. RESULTS Internalized MT1-MMP is localized at CD63-positive lysosomes. Internalization of 7 FLAG-tagged MT1-MMP (MT1F) in HeLa cells was examined by fluorescence immunostaining.MT1F was localized at the periphery of permeabilized and non-permeabilized cells at 0 time (Fig. 1A). While the signal became weaker at 30 min and...