A tandem sequence motif acts as a distance-dependent enhancer in a set of genes involved in translation by binding the proteins NonO and SFPQ

Abstract

Background: Bioinformatic analyses of expression control sequences in promoters of co-expressed or functionally related genes enable the discovery of common regulatory sequence motifs that might be involved in co-ordinated gene expression. By studying promoter sequences of the human ribosomal protein genes we recently identified a novel highly specific Localized Tandem Sequence Motif (LTSM). In this work we sought to identify additional genes and LTSM-binding proteins to elucidate potential regulatory mechanisms.

Results: Genome-wide analyses allowed finding a considerable number of additional LTSM-positive genes, the products of which are involved in translation, among them, translation initiation and elongation factors, and 5S rRNA. Electromobility shift assays then showed specific signals demonstrating the binding of protein complexes to LTSM in ribosomal protein gene promoters. Pull-down assays with LTSM-containing oligonucleotides and subsequent mass spectrometric analysis identified the related multifunctional nucleotide binding proteins NonO and SFPQ in the binding complex. Functional characterization then revealed that LTSM enhances the transcriptional activity of the promoters in dependency of the distance from the transcription start site.

Conclusions: Our data demonstrate the power of bioinformatic analyses for the identification of biologically relevant sequence motifs. LTSM and the here found LTSM-binding proteins NonO and SFPQ were discovered through a synergistic combination of bioinformatic and biochemical methods and are regulators of the expression of a set of genes of the translational apparatus in a distance-dependent manner.

Figure 1

Genome-wide analysis of LTSM. (A, B) Human transcripts from EnsEMBL55 were searched for LTSM in their promoters (-500 to +500 relative to TSS) on both strands. Histograms of the LTSM - TSS distances in the promoter set (bin width: 20 bp): (A) without prior repeat masking and (B) with prior repeat masking. (C) Number of LTSM-positive genes (LTSM between +21 to +100 bp relative to TSS of human transcripts in EnsEMBL55) without prior repeat masking subdivided into different structural classes (grey bar: forward strand, black bar: reverse-complement strand).

Figure 2

Electromobility Shift Assays (EMSAs) with LTSM-positive probes. EMSAs with LTSM-positive probes derived from eight different RP genes. The first lane contains nuclear extracts from HEK293 in the presence of labelled LTSM-positive probes. In the second lane unlabeled specific competitor (SC) probes were added to block specific protein binding. In the last three lanes unspecific competitor (UC) probes of LTSM-negative RPS6 was added. Black arrowheads indicate specific signals in five of the eight EMSAs.

Figure 3

Pull-down assays with subsequent Western blotting analysis. Biotinylated LTSM-positive probes of five RP genes were applied to nuclear extracts of HEK293 cells (lane 1 of each probe). Unspecific competitor (UC) probes derived from LTSM-negative RPS6 were applied to block unspecific competitive protein binding that often quenches the signal of the specific binding (lane 2 of each probe). In the lanes indicated with Control the procedure was performed in the absence of LTSM-positive probes. (A, B) Filled arrowheads indicate specific signals for the antibodies anti-NonO (A) and anti-SFPQ (B). Open arrowheads indicate unspecific binding of anti-NonO antibody to SFPQ (A) and anti-SFPQ antibody to NonO (B). (C) Both specific antibodies were applied in conjunction.

Figure 4

EMSA with anti-NonO and anti-SFPQ antibodies. (A) EMSA experiment using the LTSM-positive RPL36 probes and different molar excesses of specific anti-NonO and anti-SFPQ antibodies alone or in conjunction (AB). The first lane contained the labelled probe; in the following lanes nuclear extracts (NE) were added. SC indicates unlabeled specific competitor probes and UC unspecific LTSM-negative RPL13A competitor probes. Specific binding is indicated by a black arrowhead. (B) Quantitative analysis of the specific binding in EMSA experiments. Statistical relevance refers to the specific RPL36 signal (* p ≤ 0.1/** p ≤ 0.05 in two-sided t-test, n = 6).

Figure 5

Expression constructs with various LTSM - TSS distances. (A) Schema of the vector construct pRPL18: the promoter region of RPL18 containing an internal start codon (ATG) was cloned into the gene trap vector pT1β-geo in frame with the β-geo gene lacking its start codon. An Xho I restriction site was added to facilitate the insertion of linker sequences of different lengths. (B, C, D) The cells were transfected with a GFP control construct (pGFP) and the five β-geo constructs, one with the endogenous RPL18 promoter including the Xho I site (pRPL18-Xho) and four with additional linkers of the lengths 4 bp, 29 bp, 53 bp and 117 bp. (B) Northern and Western blotting analyses of β-geo mRNA and protein using a β-geo specific DNA probe and an anti-β-Galactosidase antibody in the respective experiment. (C) One representative X-Gal staining of cells transfected with the different constructs. (D) Overlay of the results of three measures of expression of the construct: mRNA, protein, and enzymatic activity (see B, C). The images were scanned and the signals quantified using ImageQuant. Northern blotting signals were normalized to the rRNA fluorescence intensities of the agarose gels (not shown) and Western blotting signals by reprobing the membranes with an anti-β-actin antibody (not shown). For each experiment the signal of pRPL18-Xho was assigned 100% and the other signals were quantified relative to it.