Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.

We have identified an easily attainable source of primitive, potentially multipotent stem cells from Wharton's jelly, the matrix of umbilical cord. Wharton's jelly cells have been propagated in culture for more than 80 population doublings. Several markers for stem cells, including c-kit (CD117), and telomerase activity are expressed in these cells. Treatment with basic fibroblast growth factor overnight and low-serum media plus butylated hydroxyanisole and dimethylsulfoxide induced Wharton's jelly cells to express a neural phenotype. Within several hours of this treatment, Wharton's jelly cells developed rounded cell bodies with multiple neurite-like extensions, similar to the morphology of neural stem cells. Neuron-specific enolase (NSE), a neural stem cell marker, was expressed in these cells, as shown by immunocytochemistry. Immunoblot analysis showed similar levels of NSE expression in both untreated and induced Wharton's jelly cells. After 3 days, the induced Wharton's jelly cells resembled bipolar or multipolar neurons, with processes that formed networks reminiscent of primary cultures of neurons. The neuron-like cells in these cultures stained positively for several neuronal proteins, including neuron-specific class III beta-tubulin, neurofilament M, an axonal growth-cone-associated protein, and tyrosine hydroxylase. Immunoblot analysis showed increasing levels of protein markers for mature neurons over time post induction. Markers for oligodendrocytes and astrocytes were also detected in Wharton's jelly cells. These exciting findings show that cells from the matrix of umbilical cord have properties of stem cells and may, thus, be a rich source of primitive cells. This study shows their capacity to differentiate into a neural phenotype in vitro.

Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribosomal subunit (SSU) processome is a large ribonucleoprotein (RNP) required for processing of precursors to the small subunit RNA, the 18S, of the ribosome. We have found that a subcomplex of SSU processome proteins, the t-Utps, is also required for optimal rRNA transcription in vivo in the yeast Saccharomyces cerevisiae. The t-Utps are ribosomal chromatin (r-chromatin)-associated, and they exist in a complex in the absence of the U3 snoRNA. Transcription is required neither for the formation of the subcomplex nor for its r-chromatin association. The t-Utps are associated with the pre-18S rRNAs independent of the presence of the U3 snoRNA. This association may thus represent an early step in the formation of the SSU processome. Our results indicate that rRNA transcription and pre-rRNA processing are coordinated via specific components of the SSU processome.

The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5 end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomalsubunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.Ribosomes are essential for the translation of mRNA into protein. Ribosome biogenesis in Saccharomyces cerevisiae begins with the transcription of the 35S pre-rRNA, which is then cleaved and processed at more than 10 different processing sites to give rise to the mature 18S, 25S, and 5.8S rRNAs (Fig. 1).Small nucleolar ribonucleoproteins (snoRNPs) are required for many of the different processing steps and modifications that occur relative to the pre-rRNA (16). There are three classes of snoRNPs (H/ACA box, C/D box, and RNase mitochondrial RNA processing) that are required for ribosome biogenesis, each of which contains a small nucleolar RNA (snoRNA). H/ACA box snoRNAs are required for site-specific pseudouridylation of rRNA, while C/D box snoRNAs are required for 2Ј-O-ribose methylation of specific nucleotides in rRNA.The U3 snoRNA and its associated proteins are required for the processing of the small ribosomal subunit at cleavage sites A 0 , A 1 , and A 2 (Fig. 1). Cleavages at A 0 and A 1 in the 5Ј external transcribed spacer mature the 5Ј end of the prerRNA. Cleavage at A 2 or A 3 in internal transcribed spacer 1 separates the small-ribosomal-subunit precursor rRNA from the large-ribosomal-subunit precursors. Defects in cleavage at the A 0 , A 1 , and A 2 sites lead to a reduction in the levels of the 18S rRNA. This reduction causes accumulation of the 35S and 23S pre-rRNAs and a reduction in the levels of the 27SA 2 and 20S pre-rRNAs (39).A large RNP required for the processing of the small-ribosomal-subunit rRNA, called the small-subunit (SSU) processome, has recently been purified (4). This preribosomal complex contains the U3 snoRNA and at least 28 proteins. We defined the SSU processome components as having the following properties. (i) They are nucleolar. (ii) They...

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