Pol I transcription and pre-rRNA processing are coordinated in a transcription-dependent manner in mammalian cells

Abstract

Pre-rRNA synthesis and processing are key steps in ribosome biogenesis. Although recent evidence in yeast suggests that these two processes are coupled, the nature of their association is unclear. In this report, we analyze the coordination between rDNA transcription and pre-rRNA processing in mammalian cells. We found that pol I transcription factor UBF interacts with pre-rRNA processing factors as analyzed by immunoprecipitations, and the association depends on active rRNA synthesis. In addition, injections of plasmids containing the human rDNA promoter and varying lengths of 18S rDNA into HeLa nuclei show that pol I transcription machinery can be recruited to rDNA promoters regardless of the product that is transcribed, whereas subgroups of pre-rRNA processing factors are recruited to plasmids only when specific pre-rRNA fragments are produced. Our observations suggest a model for sequential recruitment of pol I transcription factors and pre-rRNA processing factors to elongating pre-rRNA on an as-needed basis rather than corecruitment to sites of active transcription.

Figure 1.

UBF interacts with fibrillarin. (A) Immunoprecipitation reaction using an anti-UBF antibody in HeLa cell lysates and a Western blot against fibrillarin. (B) Immunoprecipitation reaction performed in HeLa cells transiently transfected with GFP-fibrillarin (full length). An antibody against endogenous UBF was used for the Western blot.

Figure 2.

Fibrillarin interacts with UBF in the form of a snoRNP. (A) Schematic representation of N-terminal GFP-fibrillarin fusion proteins with various truncation mutations at indicated amino acid residues. (B) Immunoprecipitations using anti-GFP antibodies in HeLa cells that were transiently transfected with a variety of constructs: beads alone (first lane, −), pEGFP-C1-Fib (second lane, FL), pEGFP-C1-Fib1 (third lane, 1), pEGFP-C1-Fib2 (fourth lane, 2), pEGFP-C1-Fib3 (fifth lane, 3), pEGFP-C1-Fib3.5 (sixth lane, 3.5), pEGFP-C1-Fib4 (seventh lane, 4), pEGFP-C1-Fib5 (eighth lane, 5), pEGFP-C1-Fib6 (ninth lane, 6), pEGFP-C1 (tenth lane, G). No plasmids were transfected into cells for lysate that was loaded into lane 11, nor was this lysate used for an immunoprecipitation. Anti-UBF (top panel), anti-Nop58 (second panel), anti-GFP (third panel), and anti-Imp4 (bottom panel) antibodies were used for detection on Western blot.

Figure 3.

The interaction between UBF and fibrillarin is dependent on the integrity of RNA. Immunoprecipitation reactions of HeLa cell lysates were performed using anti-UBF antibodies. Western blots were performed using two processing factors, fibrillarin and Nop58, as indicated. Beads alone were incubated with HeLa cell lysate as a negative control (first lane, beads alone), and HeLa cell lysate was loaded as a positive control (last lane, lysate). Anti-UBF immunopreciptiations were treated with RNase A (third lane, RNase), or the transcription inhibitor, actinomycin D (fourth lane, ActD). Band intensities were normalized against anti-UBF immunoprecipitation reactions that were untreated (second lane, untreated).

Figure 4.

rRNA transcription factors can be recruited to an injected plasmid that contains the rDNA promoter only. (A) A schematic drawing of the three plasmids used in our experiments. (B) pHr-BESΔRNA contains the rDNA promoter and no rRNA coding sequence. (C) Immunofluorescence for control, uninjected cells shows the normal nucleolar localization of the transcription factor, UBF, and pol I subunit, RPA39. (D) Both UBF and RPA39 colocalize with pHr-BESΔRNA outside of the nucleolus 2 h after injection. (E) Injection of pHr-BESΔRNA does not alter the localization of fibrillarin and Nop58, which remain within the nucleolus. Scale bar, 10 μm.

Figure 5.

rRNA transcription factors colocalize with an exogenously expressed rDNA plasmid that contains the rDNA promoter and 5′ rDNA coding sequence. (A) pHr-BES contains the rDNA promoter and 5′ rDNA coding sequence. (B) pHr-BES microinjected into HeLa nuclei colocalizes with UBF within the nucleoplasm 2 h after injection. (C) RPA39/40 is also found outside of nucleoli and colocalizes with injected pHr-BES within the nucleoplasm. Scale bar, 10 μm.

Figure 6.

Core U3 pre-rRNA processing factors colocalize to pHr-BES foci, whereas others do not. (A) Core U3 snoRNP-processing factors, fibrillarin and Nop58, localize to nucleoli in uninjected cells. (B) Fibrillarin and Nop58 are found outside nucleoli, in the nucleoplasm, 2 h after nuclei are injected with pHr-BES. The merged image was compiled without the DAPI layer and shows that fibrillarin and Nop58 colocalize with the pHr-BES speckles. (C) U3 snoRNA is recruited to the same subnuclear (D) areas as injected pHr-BES. (E) Pre-rRNA processing factors Sof1 and B23 localize to nucleoli in control cells not injected with plasmids. (F) The localization patterns of Sof1 and B23 do not change 2 h after the nuclei were injected with the plasmid. Neither Sof1 nor B23 are recruited to the pHrBES foci. Scale bar, 10 μm.

Figure 7.

The U3-associated, noncore rRNA processing factor Sof1 is recruited to a plasmid that expresses the entire 5′ ETS and a majority of the 18S gene. (A) pHrB contains the rDNA promoter, the entire 3700-base pair external transcribed spacer (ETS), and the first 1639 base pairs of the 18S rRNA. (B) The U3-associated, noncore processing factor Sof1 is recruited from nucleoli to colocalize with the injected pHrB plasmid in the nucleoplasm at 2 h after injection. By contrast, the late processing factor B23 remains entirely in the nucleoli.