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. 2017 Jul 7;45(12):7285-7298.
doi: 10.1093/nar/gkx454.

Genome-wide mRNA Processing in Methanogenic Archaea Reveals Post-Transcriptional Regulation of Ribosomal Protein Synthesis

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Free PMC article

Genome-wide mRNA Processing in Methanogenic Archaea Reveals Post-Transcriptional Regulation of Ribosomal Protein Synthesis

Lei Qi et al. Nucleic Acids Res. .
Free PMC article

Abstract

Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry.

Figures

Figure 1.
Figure 1.
Determination of the processing sites in the transcriptome of M. psychrophilus R15. (A) Flowchart for the filtration steps in identification of the authentic RNA processing sites (PSSs). On the basis of statistics described in Materials and Methods, the criterion for each filtration is indicated. RC, read count; 5΄P/+, count ratio of a site in (5΄P) to (+) library; +1/−1, count ratio of PSS to that of 1 nt upstream; PSS/TSS, count ratio of a PSS to the corresponding TSS in (−) library. (B) A diagram for PSS definition. uPSS, cPSS and iPSS represent the processing sites in the 5΄ UTR, coding region and IGR, respectively. Blue and magenta arrows indicate TSS and PSS, respectively. (C) A pie chart showing distribution of the identified PSS in the transcriptome. ORF-N, -M and -C specify the PSS location at the N-terminal, middle, and C-terminal region, respectively, of the encoding sequence.
Figure 2.
Figure 2.
Characteristic patterns of the processing sites and functional enrichment of PSS-associated genes in R15. (A) Boxplots show distance (d) distribution of uPSSs to their TSS/start codon and the 5΄ UTR lengths (left), and iPSSs to the downstream start codon/upstream stop codon and the IGR length (right). (B) A diagram shows the location distribution percentage of uPSS in 5΄ UTR and iPSS within IGR shown inside the parentheses. PSS (magenta arrow), RBS (red box) and translation start codon (light blue box) are shown. (C) The processing motif of uPSS and iPSS based on alignment of sequence around processing sites. Positions on the x-axis are relative to the PSS. (D) Distribution of uPSS- and iPSS-associated genes amongst different COGs. Note that the majority of uPSS- and iPSS-associated genes belong to four COGs: J, O, E and C.
Figure 3.
Figure 3.
Primer extension analysis of uPSS-mediated transcripts of ribosomal protein genes rpsO (A) and rps17e (B), and thermosome-related genes cpn60 (C) and tap (D). Blue and magenta arrows indicate TSSs and PSSs, respectively. RBSs are shadowed by red box. Sequencing reads of the TSS in (+) library, both TSS and PSS in (−) library and PSS only in (5΄P) library are shown in the left panels. The y-axis scale represents the read count of a given site. Primer extension gels are shown in the right panels. The numbers 18 and 8 on the gel top indicate RNA isolated from R15 cultures grown at 18°C and 8°C, respectively.
Figure 4.
Figure 4.
Primer extension and Northern blot analyses of iPSS in the rplA (A), rps19e (B), rpl7Ae (C) and leuD (D) operons. Sequencing reads of the TSS in (+) library, both TSS and PSS in (−) library and PSS only in (5΄P) library are shown in the left panels, primer extension gels are shown in the middle panels, and Northern blot gels are shown in the right panels. Magenta arrows indicate PSSs, and blue arrows indicate TSSs. iPSSs on primer extension gels are indicated as the same Arabic numerals as those in the operon diagram. Roman numerals on Northern blot gels indicate the transcripts with the expected length according to TSS and PSS. The numbers 18 and 8 on the gel top indicate RNA isolated from R15 cultures grown at 18°C and 8°C, respectively. The 23S and 16S rRNA bands in (A) were due to cross hybridization with the probe used.
Figure 5.
Figure 5.
5΄ UTR processing stabilizes the transcript of the ribosomal protein gene rpsO and activates its translation. (A, D) Northern blot or primer extension probes the stabilities of PSS-mediated mRNA isoforms of rpsO and rps17e in R15 cells. The 5΄ UTR lengths for primary and processed mRNA isoforms are shown to the right of the gel (upper panel). Half-lives are calculated from the regression curve of mRNA remnant (lower panel). (B, E) 5΄ UTR secondary structures of rpsO and rps17e transcripts predicted by Mfold. Red and light blue letters show RBS and translation start codon, respectively. Magenta and red arrows indicate the PSSs. (C, F) Translation efficiencies of mRNA isoforms of rpsO and rps17e with different lengths of 5΄ UTR by processing. A schematic diagram shows the construction of lacZ translational fusion (upper panel). A LB plate containing X-gal visualizes the expression of lacZ (left), and a histogram shows the β-galactosidase activities upon IPTG induction (right). The β-galactosidase activities are the mean determination of three reporter strains, and standard deviations are shown.
Figure 6.
Figure 6.
IGR processing within the tricistronic operon rplA-rplJ-rplL enhances rplL translation and tunes the stoichiometric ratio of L10 and L12. (A) A diagram shows gene organization of the tricistronic operon rplA-rplJ-rplL with two iPSSs within the IGR of rplJ-rplL (upper panel), and the half-lives of the two transcripts calculated from an unpublished transcriptomic data (lower panel). (B) Mfold predicts the secondary structures within the IGR of rplJ-rplL dicistronic transcript. Red and light blue letters show RBS and translation start codon, respectively. Magenta arrows indicate the PSSs. (C) Translation efficiencies of iPSSs-mediated mRNA isoforms of rplL. Interpretations for the diagrams and X-gal plate are the same as for Figure 5C. (D) Western blot analyses determine the protein ratio of L12 to L10 to be 4:1. Purified proteins L12 and L10 are loaded with the indicated contents on SDS-PAGE gel, and by using the antibodies protein content calibrations are determined for L12 and L10, respectively (left). The cellular L12 and L10 levels (ng) are determined at an OD600 of 0.4, and the protein molecules are calculated based on the calibrations (right). (E) mRNA abundance of rplJ and rplL in R15 at different temperatures. The fold changes of rplL to rplJ are shown above the histogram of rplL.
Figure 7.
Figure 7.
Growth-associated mRNA processing and abundance of ribosomal proteins. (A) Northern blot detects the primary and processed transcripts of rpsO during growth. OD600 0.2, 0.4, 0.6, and 0.66 correspond respectively to the early, middle, late exponential, and stationary growth phases referenced in Supplementary Figure S7. Primary (blue arrow) and processed (magenta arrows) transcripts are indicated and 7S RNA serves as internal standard. (B) Histogram of transcripts level of rpsO primary and processed isoforms. (C) The cellular protein level of S15 shown as Western blot bands (upper panel) and quantitative analysis displayed by histogram (lower panel). (D) Northern blot probe of the iPSS-mediated processed transcript of rplL and the tricistronic primary transcript (left). (E) Histogram of transcripts level of rplL primary and processed isoforms. (F) Western blot displaying the protein expression level of L10 and L12 during growth (upper panel) and quantitative analysis of the stoichiometric ratio of L10:L12 (lower panel).
Figure 8.
Figure 8.
A schematic model of RNA processing involved in the post-transcriptional regulation of the methanogenic archaeal ribosomal protein synthesis and stoichiometry. The methanogenic archaeal genes or operons that encode ribosomal and other housekeeping proteins frequently possess a long 5΄ UTR or IGR that form complex RNA structures. RNA processing removes the bulk structure and liberates the RBS. Processing of the 5΄ UTRs enhances translation initiation, while processing of the IGRs would tune the proportional translation of the co-transcribed transcripts, thus harness the stoichiometry of the hetero-multiprotein complex like ribosome. Efficient translation also stabilizes the processed transcripts. The blue and magenta arrows specify TSS and PSS, respectively. Red box indicates RBS and Ter represents transcription terminator.

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