Interactions between the 5' UTR mRNA of the spe2 gene and spermidine regulate translation in S. pombe

Abstract

The 5' untranslated regions (5' UTR) of mRNAs play an important role in the eukaryotic translation initiation process. Additional levels of translational regulation may be mediated through interactions between structured mRNAs that can adopt interchangeable secondary or tertiary structures and the regulatory protein/RNA factors or components of the translational apparatus. Here we report a regulatory function of the 5' UTR mRNA of the spe2 gene (SAM decarboxylase) in polyamine metabolism of the fission yeast Schizosaccharomyces pombe Reporter assays, biochemical experiments, and mutational analysis demonstrate that this 5' UTR mRNA of spe2 can bind to spermidine to regulate translation. A tertiary structure transition in the 5' UTR RNA upon spermidine binding is essential for translation regulation. This study provides biochemical evidence for spermidine binding to regulate translation of the spe2 gene through interactions with the 5' UTR mRNA. The identification of such a regulatory RNA that is directly associated with an essential eukaryotic metabolic process suggests that other ligand-binding RNAs may also contribute to eukaryotic gene regulation.

Keywords: regulatory RNA; spermidine.

FIGURE 1.

A brief illustration of polyamine biosynthesis pathway. Dark blue indicates the regulated decarboxylases, the polyamine biosynthesis pathway is shown in light blue, and S-adenosyl methionine metabolites are shown in green.

FIGURE 2.

Spd control of reporter gene expression through the 5′ UTR of spe2. (A) The lacZ reporter constructs, incorporating the 5′ UTRs of spe2, aru1, srm1, and sam1. (B) β-gal expression in the presence of 0, 10, and 1000 µM of Spd with (±) thiamine in Δspe2 cells transformed by empty reporter vector or the spe2 5′ UTR reporter plasmid. Statistical significance was calculated compared to controls using Student's t-test, (**) P < 0.01. The error bars are the standard deviation of three independent experiments. (C) β-gal expression measured in response to increasing doses of Spd without thiamine in Δspe2 cells that were transformed with the reporter plasmid containing the 5′ UTRs, as listed in A. Statistical significance was calculated compared to other controls using Student's t-test, (**) P < 0.01. The error bars are the standard deviation of three independent experiments. (D) The GFP reporter constructs with the 5′ UTRs of spe2. (E) Reporter activity of Δspe2 cells transformed with the spe2 5′ UTR GFP reporter plasmid, in response to 10 µM of Spd with (+) and without (−) thiamine (thi). GFP was detected by western blot. GFP expression is enhanced approximately fivefold by adding 10 µM of Spd, relative to no Spd normalized against actin.

FIGURE 3.

The polyamines bind to the spe2 UTR RNA. (A) Change in SPR signal in response units (RU) on Spd bound to immobilized spe2 UTR RNA. Hill coefficient is shown. (B) Change in SPR signal in RU on Spm bound to immobilized spe2 UTR RNA. Hill coefficient is shown. (C) The relationship between polyamine structure, binding affinity, and reporter gene induction. NB represents no binding.

FIGURE 4.

Secondary structure of the 5′ UTR of spe2 RNA. (A) The structure of the crosslinker LC-SDA. (B) Electropherogram of a photoactive DA-Spd and RNA crosslinks. The in vitro transcribed RNA was irradiated with UV light with DA-Spd (blue), compared with the in vitro transcribed RNA irradiated with DA-Spd, and a 10-fold excess of unlabeled Spd (red) and the in vitro transcribed RNA irradiated with inactive DA-Spd (green) as controls. (C) Electropherogram of DMS-modified RNA with DMS ([+] DMS, blue trace), compared with that of unmodified RNA ([−] DMS, gray trace). (D) Predicted secondary structure of the 5′ UTR RNA by Vienna computational folding, covariance modeling, and chemical probing. Black circles filled with green indicate the DMS reactive nucleotides, and red indicates the crosslinking nucleotides.

FIGURE 5.

OsO₄ probing of tertiary structure of spe2 RNA on polyamine titration. (A) Analysis of OsO₄ modification electropherogram of RNA with 30 µM Spd (blue trace), compared with 0 µM Spd (red trace). (B) Histogram of OsO₄ modification of the in vitro transcribed RNA nucleotides (58–62) on titration of Spd. The reactivity of the nucleotide is calculated as the ratio of the height of the nucleotide to the height of all nucleotides. (C) Fitting curve for the ratio of OsO₄ modification signal of position U59 to that of full length on titration of Spd; KD is shown.

FIGURE 6.

Functional and structural specificity of Spd binding to the spe2 UTR RNA. (A) Real-time PCR analysis of mRNA abundance of the spe2 UTR RNA relative to the internal control tubulin, showing the level of mRNA abundance of lacZ (representing spe2) remains unchanged on Spd titration. Error bars represent the standard deviation of three independent experiments. Statistical significance was shown compared with 0 µM, respectively, using Student's t-test, (**) P < 0.01. (B) The positions (boxed dark blue) and identity of the mutations on the secondary structure of the spe2 UTR RNA. The position boxed in purple indicates the mutation of the initiation codon of the uORF. (C) Reporter gene expression of seven inactive RNA mutations, responding to 0, 10, and 1000 µM of Spd. The error bars are the standard deviation of at least three independent experiments. Statistical significance of each mutant was calculated compared to that of WT at 0, 10, and 1000 µM, respectively, using Student's t-test, (**) P < 0.01. (D) Reporter gene expression of seven inactive RNA mutations at 0 µM of Spd. The error bars are the standard deviation of at least three independent experiments. Statistical significance of each mutant was calculated compared to that of WT at 0 µM, respectively, using Student's t-test, (**) P < 0.01. (E) Reporter gene expression of inactive RNA mutations M25–26, M28, M30, M32, M34, and M36–37, responding to 0, 10, and 1000 µM of Spd. The error bars are the standard deviation of at least three independent experiments. Statistical significance of each mutant was calculated compared to that of WT at 0, 10, and 1000 µM, respectively, using Student's t-test, (**) P < 0.01. (F) Histogram analysis of OsO₄ modification for nucleotides U59 of the wild-type and three inactive mutation RNAs on Spd titrations.

FIGURE 7.

The uORFs in the spe2 5′UTR have no dominant translational function. (A) The locations of the micro-ORF in the spe2 UTR; gray indicates peptide sequence, and black rectangle filled with yellow indicates start codon mutation M6 and M22 in the peptide. (B) Sequences of the wild-type peptide and the corresponding RNAs, M6 start codon mutant and their degenerate mutants M42–45 (mutations marked pink). The peptide RNA sequences of M42–45 were altered, whereas the peptide amino acid sequences remained unchanged. The sequences of the M22 start codon mutant are also shown. (C) β-gal activity measured in the presence of 0, 10, and 1000 µM of Spd without thiamine in Δspe2 cells that were transformed with the reporter plasmid containing mutants M6, M22, and M42–45. Statistical significance was calculated compared to WT at 0, 10, and 1000 µM, respectively, using Student's t-test, P < 0.01.