RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity

Abstract

During recent studies of ribonucleolytic ''degradosome'' complexes of Escherichia coli, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is ssrA (for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt ssrA RNA is generated by RNase E cleavage at the CCA-3 terminus of a 457-nt ssrA RNA precursor and that interference with this cleavage in vivo leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature ssrA RNA and for normal ssrA RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in E. coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3 end of ssrA RNA. R ibonuclease E (RNase E), which is essential for cell growth and was initially characterized as the enzyme that processes 9S RNA to yield a 5S product, p5S RNA (1), has been shown to control the rate-limiting step in the degradation of a variety of Escherichia coli mRNAs (for reviews, see refs. 2-5), and a small regulatory RNA, RNAI-an antisense repressor of the replication primer of ColE1-type plasmids (6 -8). Temperaturesensitive mutants of RNase E have been isolated (9-12) and mapped to its enzymatic active domain (13), which is located at the N-terminal half of the polypeptide (14, 15). Inactivation of RNase E activity in these mutants by culture at nonpermissive temperature prolongs the decay of bulk mRNA (11,12).Recently, an RNase E-containing multicomponent ribonucleolytic complex termed the ''RNA degradosome'' has been isolated and characterized by two distinct approaches (16, 17), both of which show that degradosomes contain the protein components RNase E, PNPase, RhlB RNA helicase, and enolase. In addition to these proteins, the degradosome was found to contain the chaperonins DnaK (16, 18) and GroEL (16), polyphosphate kinase (18), mRNA, and certain structural RNA species (16,19), including RNAI, fragmented rRNAs, 9S RNA, and 10Sa͞ssrA RNA [a small stable RNA encoded by the ssrA gene (20)(21)(22)]. Most of these RNAs are known to be substrates for RNase E (19,[23][24][25][26].Nascent polypeptides translated in E. coli from truncated mRNAs lacking stop codons commonly receive a short carboxylterminal peptide tag (27, 28), synthesized in trans (28,29), that targets the resulting fusion polypeptide for rapid degradation (28). Except for the first alanine, the amino acid sequence of this tag (AANDENYALAA) is encoded by 10Sa͞ssrA RNA (20)(21)(22), also known as tmRNA because of its dual tRNA-like and mRNA-like activities (22,(28)(29)(30). Mature 363-nt ssrA RNA, which has its 3Ј and 5Ј ends paired in a tRNA-like structure, is charged with an alanine residue (22, 30), ...

Scarce adenylation in bacteriophage T4 mRNAs.

“…Since the degradation of T4 mRNA in a stage-dependent manner would require timely and quantitative expression of genes at former stages (Kai et al, 1998;Ueno and Yonesaki, 2001), it may be possible that an rne mutation affects T4 mRNA degradation via gene expression. Furthermore, RNase E also takes a part in processing of various functional RNAs such as ribosomal RNAs (Ghora and Apirion, 1978;Li et al, 1999;Wachi et al, 1999), tRNAs (Li and Deutscher, 2002;Ow and Kushner, 2002), the RNA subunit of RNase P (Lundberg and Altman, 1995) and ssrA RNA (Lin-Chao et al, 1999), all of which are essential for normal activity of protein synthesis and its quality control. Therefore, it is not clear whether the effect of RNase E on T4 mRNA turnover is direct or indirect.…”

Mycobacterial RNase E‐Associated Proteins

“…Subsequent studies revealed that RNase E controls the stability of many (8, 9), if not most, mRNAs in Escherichia coli (3), as well as being involved in the processing of 5S and 16S rRNAs (1,20,32), tRNA precursors (19) and degradation of small regulatory RNAs (21). Moreover, RNase E has the ability to affect protein degradation through its role in maturation of the 3' end of tmRNA (SsrA) (22).Purification studies have revealed that in E. coli RNase E forms a large multienzyme complex referred to as 'degradosome ' (5, 24, 26). In addition to RNase E, the other major components of this complex are polynucleotide phosphorylase (pnpase), enolase and RhlB RNA helicase.…”

“…Subsequent studies revealed that RNase E controls the stability of many (8, 9), if not most, mRNAs in Escherichia coli (3), as well as being involved in the processing of 5S and 16S rRNAs (1,20,32), tRNA precursors (19) and degradation of small regulatory RNAs (21). Moreover, RNase E has the ability to affect protein degradation through its role in maturation of the 3' end of tmRNA (SsrA) (22).…”

DEAD Box RhlB RNA Helicase Physically Associates with Exoribonuclease PNPase to Degrade Double-stranded RNA Independent of the Degradosome-assembling Region of RNase E

“…[5][6][7][8][9][10][11]. The multicomponent complex consists of: the RNA endonuclease RNase E, whose activity is essential for Escherichia coli cell growth (12)(13)(14), RNA processing (15,16), and degradation (17,18); the 3Ј-5Ј exoribonuclease PNPase (19); RhlB RNA helicase (20); and enolase (21), an enzyme involved in the glycolytic pathway and other chaperonin proteins (3,22). Interestingly, in addition to mRNAs, highly structured, stable RNA fragments have also been found to be associated with RNA degradosome complexes (3,23), which implies quality control by the RNA degradosome for the biogenesis of stable RNAs.…”