The splice variants of UBF differentially regulate RNA polymerase I transcription elongation in response to ERK phosphorylation

Abstract

The mammalian architectural HMGB-Box transcription factor UBF is ubiquitously expressed in two variant forms as the result of a differential splicing event, that in the UBF2 deletes 37 amino acid from the second of six HMGB-boxes. Several attempts to define a function for this shorter UBF2 protein have been less than satisfactory. However, since all mammals appear to display similar levels of the longer and shorter UBF variants, it is unlikely that UBF2 is simply nonfunctional. Previously we showed that phosphorylation of UBF by the MAP-kinase ERK regulates chromatin folding and transcription elongation, explaining the rapid response of the ribosomal RNA genes to growth factors. Here we have investigated the roles the UBF variants play in the response of these genes to ERK activity. We demonstrate that the variant HMGB-box 2 of UBF2 has lost the ability to bind bent DNA and hence to induce chromatin folding. As a result it is significantly less effective than UBF1 at arresting RNAPI elongation but at the same time is more responsive to ERK phosphorylation. Thus, UBF2 functionally simulates a hemi-phosphorylated UBF whose expression may provide a means by which to tune the response of the ribosomal RNA genes to growth factor stimulation.

Figure 1.

(A) The domain structure of the mammalian UBF splice variants. The HMGB-Box domains are shown using HMGD as model (27). The UBF2 splice variation is shown as a hypothetical folded structure and the variant polypeptide indicated in green. The asterisk indicates the ERK phosphorylation sites and ‘S’ the serine rich segment in the acidic ‘tail’ domain (zig-zag line). (B) The protein sequence of full length HMGB-Box 2.1 and shortened Box2.2 showing the effect of differential UBF mRNA splicing. Predicted alpha-helical regions are shown in red and asterisk indicates the ERK phosphorylation site.

Figure 2.

UBF1 and UBF2 differ in their abilities to inhibit RNAPI transcription during multiple rounds of initiation. (A) Baculovirus expressed full-length rat UBFs. A Commassie stained gel of increasing amounts of the two proteins is shown. (B) In vitro RNAPI transcription in the presence of increasing amounts of the full-length UBFs. The phosphoimage of a typical electrophoretic gel analysis is shown and below this the quantitation of this analysis. (C) Typical time course of in vitro transcript accumulation in the presence or absence of 1200 ng per standard reaction of UBF1 or 2. As in B, quantitation of the analysis is shown below the phosphoimage of the gel, the right-hand panel shows a Y-axis expansion. Error bars indicate estimated measurement errors in B and C.

Figure 3.

UBF1 arrests elongation by RNAPI transcription complexes more efficiently than UBF2. (A) Diagram delineating the experimental procedure to measure elongation efficiency. Transcription of the RNAPI template containing a G-less cassette, bases 1 and 34, was initiated by the addition of the DEAE 280 nuclear protein fraction (DEAE fraction, see ‘Materials and methods’ section) in the presence of ATP, CTP and α[³²P]-UTP. One thousand two hundred nanograms of UBF was added to the RNAPI elongation complexes arrested at +34 and finally GTP and excess UTP unlabeled was added to permit elongation to continue to the end of the template. (B) Typical phosphoimage analysis of a G-less cassette transcription assays. For tracks in which full-length rat UBF1 or 2 were added to the reactions, the amounts from left to right were 100, 300, 600, 900 and 1200 ng. ‘34b’ refers to the transcripts from elongation complexes arrested at the end of the G-less cassette and ‘320b’ to the full-length ‘run-off’ transcripts after addition of GTP (and excess UTP). Endogenous 5S RNA was also labeled during the reaction (10). (C) Quantitation of the yield of full-length (320b) transcript in the analysis shown in B as a function of UBF1 or 2 addition. Error bars indicate estimated measurement errors.

Figure 4.

The DNA architectural core regions of UBF1 and 2 emulate the activities of the full-length proteins. (A) Commassie stained gel analysis of mouse core UBF (cUBF) 1 and 2 proteins. (B) Time course of transcript yield during multi-round transcription reactions in the presence of 600 ng of cUBF1 or cUBF2. A typical analysis is shown above the quantitation. The lower right hand panel shows a Y-axis expansion of the data. (C) Elongation assays on the G-less cassette template in the presence of increasing amounts (100, 300 and 600 ng) of cUBF1 or 2. Lower panel gives the mean of three experiments and the standard errors. (D) Typical time course of elongation in the presence of 600 ng of cUBF1 or 2 for increasing GTP (+UTP) chase times. Upper panel shows the phosphoimage and lower panel the quantitation. Error bars indicate estimated measurement errors. The experimental design and nomenclature are as in Figure 3A. In B and D the curves show the best fits to the function; a + b[1−(exp−ct)], where t = time.

Figure 5.

The short HMGB-Box2.2 splice variant does not recognize prebent DNA. (A) Upper panel shows the Coomassie stained SDS–PAGE analysis of recombinant HMGB-Box1 protein, and the longer and shorter protein variants of HMGB-Box2, respectively Box2.1 and Box2.2. The lower panel shows the analysis of in vitro phosphorylation of the Boxes catalyzed by active ERK2. The percent stoichiometric levels of phosphorylation of each Box are indicated (% Stoichiometric Phosphorylation). (B) and (C) Mobility shift analyses of the binding of increasing amounts of the unphosphorylated Boxes to the standard cruciform DNA structure (9,19).

Figure 6.

UBF2 is more responsive to ERK phosphorylation. Core UBF1 and 2 (cUBF1, 2) were phosphorylated by ERK2 for increasing times or mock phosphorylated and then assayed for their abilities to arrest RNAPI elongation in the G-less cassette reaction. (A) Shows a phosphoimage of a typical gel electrophoretic analysis and (B) the quantitation of elongation efficiency plotted as full-length transcript yield as a function of time of phosphorylation and as a function of phosphorylation. Error bars indicate estimated measurement errors.

Figure 7.

Comparison of the domain structures of variant UBFs from four animal species. Orthologous HMGB-Box domains are indicated by a common coloring. In the case of Zebrafish (Danio rerio) the diagrams present only the major variants, in fact at least five distinct variants are expressed from two genes. In the case of Pufferfish (Takifugu rubripes) two distinct genes were identified but no cDNA data was available from which to determine possible splice variants. The diagrammatic representations are based on the following UBF sequences; Rat P25977, Xenopus laevis CAA40487 and CAA42523, Pufferfish (Takifugu rubripes) Ensembl peptides SINFRUP00000177953 and SINFRUP00000181780, and Zebrafish (Danio rerio) Ensembl peptides ENSDARP00000048724 and ENSDARP00000056633.