Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein-protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements.[Keywords: RNA polymerase I; ribosomal DNA; nucleolar organiser region; nucleolus; upstream-binding factor] Supplemental material is available at http://www.genesdev.org.

Efficient ribosome biogenesis requires coordination of a highly complex series of events. Early events include pre-RNA transcription, processing, and modification. Analysis in yeast has demonstrated that t-UTPs, components of the U3 snoRNA-containing pre-rRNA processing complex, are required for efficient transcription of ribosomal genes (rDNA) by RNA polymerase I (pol I). Here, we characterize human t-UTPs and establish that their ability to link transcription and pre-rRNA processing is evolutionarily conserved. The pol I transcription factor UBF binds extensively across rDNA throughout the cell cycle, resulting in a specialized form of chromatin that is the hallmark of active nucleolar organizer regions (NORs). Transcriptionally silent pseudo-NORs are ectopic, chromosomally integrated, artificial arrays that mimic this specialized chromatin structure. Pseudo-NORs sequester t-UTPs and factors linking transcription with pre-rRNA modification (Nopp140 and Treacle). Recruitment is independent of transcription, the underlying DNA sequence, and location within the nucleolus. Previously, we have demonstrated that pseudo-NORs sequester every component of the pol I transcription machinery. Taken together, these results highlight the importance of the specialized chromatin structure at active NORs in coordinating early events in ribosome biogenesis and nucleolar formation.[Keywords: Nucleolus; rDNA transcription; pre-rRNA processing; nucleolar organizer region (NOR); t-UTPs; UBF] Supplemental material is available at http://www.genesdev.org.

The fundamental process of ribosome biogenesis requires hundreds of factors and takes place in the nucleolus. This process has been most thoroughly characterized in baker's yeast and is generally well conserved from yeast to humans. However, some of the required proteins in yeast are not found in humans, raising the possibility that they have been replaced by functional analogs. Our objective was to identify non-conserved interaction partners for the human ribosome biogenesis factor, hUTP4/Cirhin, since the R565W mutation in the C-terminus of hUTP4/Cirhin was reported to cause North American Indian childhood cirrhosis (NAIC). By screening a yeast two-hybrid cDNA library derived from human liver, and through affinity purification followed by mass spectrometry, we identified an uncharacterized nucleolar protein, NOL11, as an interaction partner for hUTP4/Cirhin. Bioinformatic analysis revealed that NOL11 is conserved throughout metazoans and their immediate ancestors but is not found in any other phylogenetic groups. Co-immunoprecipitation experiments show that NOL11 is a component of the human ribosomal small subunit (SSU) processome. siRNA knockdown of NOL11 revealed that it is involved in the cleavage steps required to generate the mature 18S rRNA and is required for optimal rDNA transcription. Furthermore, abnormal nucleolar morphology results from the absence of NOL11. Finally, yeast two-hybrid analysis shows that NOL11 interacts with the C-terminus of hUTP4/Cirhin and that the R565W mutation partially disrupts this interaction. We have therefore identified NOL11 as a novel protein required for the early stages of ribosome biogenesis in humans. Our results further implicate a role for NOL11 in the pathogenesis of NAIC.

Eukaryotic 18S rRNA processing is mediated by the small subunit (SSU) processome, a machine comprised of the U3 small nucleolar RNP (U3 snoRNP), tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes. We report that the human SSU processome is a dynamic structure with the recruitment and release of subcomplexes occurring during the early stages of ribosome biogenesis. A novel 50S U3 snoRNP accumulated when either pre-rRNA transcription was blocked or the tUTP proteins were depleted. This complex did not contain the tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes but was associated with the RNA-binding proteins nucleolin and RRP5 and the RNA helicase DBP4. Our data suggest that the 50S U3 snoRNP is an SSU assembly intermediate that is likely recruited to the pre-rRNA through the RNA-binding proteins nucleolin and RRP5. We predict that nucleolin is only transiently associated with the SSU processome and likely leaves the complex not long after 50S U3 snoRNP recruitment. The nucleolin-binding site potentially overlaps that of several other key factors, and we propose that this protein must leave the SSU processome for pre-rRNA processing to occur.In eukaryotes, the 18S, 5.8S, and 28S (25S in yeast) ribosomal RNAs (rRNAs) are transcribed as a single precursor molecule by RNA polymerase I that is processed by both endoand exonucleolytic cleavages (12, 17) in the nucleolus. The production of each ribosome requires 80 ribosomal proteins and more than 150 additional factors (including exonucleases, endonucleases, chaperones, helicases, annealing factors, and small nucleolar RNPs [snoRNPs]). Ribosome biogenesis is a major consumer of energy in the cell (36); is regulated relative to development, cell growth, the cell cycle, and stress; and is upregulated in the majority of transformed cells (33).The production of 18S rRNA involves the removal of the 5Ј external transcribed spacer (5ЈETS) and internal transcribed spacer 1 by cleavages at sites AЈ, A 1 , and A 2 . This is mediated by the SSU processome, a complex that contains the U3 snoRNA and more than 40 additional proteins (reviewed in references 12 and 17). In higher eukaryotes, the nucleolus contains three subcompartments, namely, the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC) (18, 31). Pre-rRNA transcription occurs on the border between the FC and DFC, and processing intermediates migrate through the nucleolar compartments in vectorial fashion (28). The initial cleavages in the 5ЈETS (AЈ) and 3ЈETS occur in the DFC, while the removal of the core 5ЈETS sequence (A 1 cleavage) takes place subsequently in the GC. The U3 snoRNP is found throughout the nucleolus and cycles between the DFC and the GC as part of the SSU processome (8,15). Indeed, the ability of the U3 snoRNA to localize to the GC correlates with its ability to be recruited to the SSU processome and to participate in the AЈ cleavage event (15).The U3 snoRNP is present in the cell as a 12S monoparticle comprised of the U3 snoRNA, the core box C/D snoRNP proteins (15.5K [...

We have characterized the two families of SINE retroposons present in Arabidopsis thaliana. The origin, distribution, organization, and evolutionary history of RAthE1 and RAthE2 elements were studied and compared to the well-characterized SINE S1 element from Brassica. Our studies show that RAthE1, RAthE2, and S1 retroposons were generated independently from three different tRNAs. The RAthE1 and RAthE2 families are older than the S1 family and are present in all tested Cruciferae species. The evolutionary history of the RAthE1 family is unusual for SINEs. The 144 RAthE1 elements of the Arabidopsis genome cannot be classified in distinct subfamilies of different evolutionary ages as is the case for S1, RAthE2, and mammalian SINEs. Instead, most RAthE1 elements were probably derived steadily from a single source gene that was maintained intact and active for at least 12-20 Myr, a result suggesting that the RAthE1 source gene was under selection. The distribution of RAthE1 and RAthE2 elements on the Arabidopsis physical map was studied. We observed that, in contrast to other Arabidopsis transposable elements, SINEs are not concentrated in the heterochromatic regions. Instead, SINEs are grouped in the euchromatic chromosome territories several hundred kilobase pairs long. In these territories, SINE elements are closely associated with genes. A retroposition partnership between Arabidopsis SINEs and LINEs is proposed.