Fidelity of DNA and protein synthesis is regulated by a proofreading mechanism but function of a similar mechanism during RNA synthesis has not been demonstrated. Analysis of transcriptional fidelity and its control has been hampered by the necessity to employ complex DNA templates requiring either a promoter and initiation factors or 3-extended templates. To circumvent this difficulty, we have created an RNA-DNA dumbbell template that can be recognized as a template-primer and extended by RNA polymerase II. By employing this system, we demonstrate that RNA polymerase II can misincorporate a nucleotide and carry out template-dependent elongation at the mispaired end. The transcripts containing misincorporated residues can be cleaved by the very slow 3 3 5 ribonuclease activity of the RNA polymerase II, but enhancement of this activity by the elongation factor TFIIS generates RNA with a high degree of fidelity. This enhanced preferential cleavage of misincorporated transcripts suggests an important role for TFIIS in maintaining transcriptional fidelity.Maintenance of fidelity in macromolecular information transfer is important for precise replication and differentiation of an organism. DNA is replicated with astounding precision (10 Ϫ9to 10 Ϫ10 errors per base replicated); the high fidelity is achieved by a DNA polymerase capable of both base-pair discrimination and proofreading (1). The analogous question of how transcriptional fidelity might be maintained remains largely unanswered. Maintenance of fidelity during elongation is likely to be an important target for gene regulation in eukaryotes. As has been shown for Escherichia coli, the elongation͞cleavage factor GreA controls the fidelity of RNA polymerase transcription (2-4). Several eukaryotic elongation factors, TFIIF (5), TFIIS (6-10), P-TEFb (11), and elongin SIII (12), have been shown to enhance the ability of RNA polymerase II (pol II) to produce full-length mRNA transcripts, but their role in transcriptional fidelity has not yet been explored. The recent discovery that pol II harbors an intrinsic 3Ј 3 5Ј ribonuclease activity that can be potentiated by TFIIS (9, 10, 13, 14) led us to investigate the role of this factor in transcriptional fidelity.Assessment of transcriptional fidelity has been impeded by the difficulties in generating functional elongation complexes halted at specific sites and in precise analysis of the RNA products. While promoter-dependent transcription by E. coli RNA polymerase of a DNA template containing PR promoter allowed study of the fidelity of transcription and its control by elongation factor GreA, these studies are complex and require sophisticated kinetic analysis to resolve the data (2). Other templates not requiring any accessory factors such as 3Ј-extended DNAs and the synthetic RNA-DNA bubble duplexes can be accurately initiated and elongated by pol II but they also pose difficulties in simple biochemical analysis of the RNA products to identify misincorporation (7,15).In the present studies, we have designed ...

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Human mesenchymal stromal cells (MSCs) offer great hope for the treatment of tissue degenerative and immune diseases, but their phenotypic similarity to dermal fibroblasts may hinder robust cell identification and isolation from diverse tissue harvests. To identify genetic elements that can reliably discriminate MSCs from fibroblasts, we performed comparative gene and microRNA expression profiling analyses with genome-wide oligonucleotide microarrays. When taken globally, both gene and microRNA expression profiles of MSCs were highly similar to those of fibroblasts, accounting well for their extensive phenotypic and functional overlaps. Scattered expression differences were pooled to yield an MSC-specific molecular signature, consisting of 64 genes and 21 microRNAs whose expressions were at least 10-fold and two-fold higher, respectively, in MSCs compared with fibroblasts. Genes either encoding transmembrane proteins or associated with tumors were relatively abundant in this signature. These data should provide the molecular basis not only for the discovery of novel diagnostic markers discriminating MSCs from fibroblasts, but also for further studies on MSC-specific signaling mechanisms.

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Summary Mesenchymal stromal cells (MSCs) have gained widespread popularity in cell therapy, but their development into clinical products has been impeded by the scarcity of cell‐specific markers. We previously explored transcriptome and membrane proteome of MSCs, from which fibroblast activation protein α (FAP) was recognized as a prime surface marker candidate. The present study showed that FAP was constitutively expressed on MSCs, but not on other cells. FAP immunoselection yielded homogeneous MSCs from cryopreserved bone marrow (BM). These results suggest that FAP serves as a surface protein marker that can singly define MSCs from BM and possibly from other sources.