RNase E-based degradosome modulates polyadenylation of mRNAs after Rho-independent transcription terminators in Escherichia coli

Abstract

Here we demonstrate that the RNase E-based degradosome is required for poly(A) polymerase I (PAP I)-dependent polyadenylation after Rho-independent transcription terminators for both mono- and polycistronic transcripts. Disruption of degradosome assembly in mutants lacking the polynucleotide phosphorylase (PNPase) binding domain led to a significant increase in the level of PNPase synthesized polynucleotide tails in the rpsJ and rpsM polycistronic transcripts and the lpp monocistronic transcript. The polynucleotide tails were mostly located within the coding sequences in the degradosome mutants compared to the wild type control where the majority of the PAP I synthesized poly(A) tails were after the Rho-independent transcription terminators. For the Rho terminated metNIQ operon, the tails for all three mRNAs were predominately polynucleotide and were located within the coding sequences in both wild type and degradosome mutant strains. Furthermore, by employing a pnp-R100D point mutant that encodes a catalytically inactive PNPase protein that still forms intact degradosomes, we show that a catalytically active PNPase is required for normal mRNA polyadenylation by PAP I. Our data suggest that polyadenylation requires a functional degradosome to maintain an equilibrium between free PNPase and the PAP I polyadenylation complex.

Figure 1

Graphical presentation of the rpsJ (also known as the S10 operon) (A), rpsM (B) and metN operons (C). The lack of spacers indicate translational overlap between specific ORFs.

Figure 2

Graphical presentation of poly(A) and polynucleotide tails found on (A) rpsQ and (B) lpp transcripts in various genetic backgrounds. Poly(A): poly(A) tail, Poly(N): polynucleotide tail. The numbers in each row of the table represent how many poly(A) or polynucleotide (PN) tails were obtained in that particular region from the corresponding genetic background. Independent isolates were sequenced as described in the text.

Figure 3

Northern analysis of the rpsJ primary transcript. Total RNA was isolated and separated on a 1.5% glyoxal agarose gel as described in the Experimental Procedures. Lane 1, MG1693; lane 2, SK2538; lane 3, SK5665; lane 4, SK9795. The sizes of the various transcripts are indicated on the left of the blot and are based on a RNA sizing ladder (Riboruler^™, Fermentas). Lanes that were not relevant were removed from the blot. The blot was scanned using a STORM 840 PhosphorImager (GE Healthcare) and the intensities of the bands were quantified using ImageQuant TL 5.2 software. The relative quantity (RQ) of the full-length transcript compared to the MG163 control is shown below each lane and represents the average of three independent determinations.

Figure 4

Graphical representation of the RNase E degradosome mutants used in this study. The catalytic region of amino acids 1-529 and the degradosome scaffold region of 530-1061 amino acids are shown. The degradosome scaffold region includes the binding sites for the RhlB RNA helicase, enolase, Hfq and PNPase (Vanzo et al., 1998, Ikeda et al., 2011, Ait-Bara et al., 2015).

Figure 5

Poly(A) sizing assay for various mutants. Total RNA from various strains was 3′-end-labeled with [³²P]-pCp, digested, and then separated on a 20% polyacrylamide gel as described in the Experimental Procedures. The genotype of each strain is listed above each lane. Lane 1, A 5′-[³²P]-end-labeled oligo(A) ladder (nt).