In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3' and 5' termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5' maturation of 16S rRNA is completely blocked. In contrast, 3' maturation is essentially unaffected. The 5' unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5' end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G.

Mutations within PCSK9 (proprotein convertase subtilisin/ kexin type 9) are associated with dominant forms of familial hyper-and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an ϳ150-fold higher affinity at an acidic endosomal pH (K D ‫؍‬ 4.19 nM) compared with a neutral pH (K D ‫؍‬ 628 nM). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.PCSK9 (proprotein convertase subtilisin/kexin type 9) encodes the ninth member of the mammalian proprotein convertase family of serine endoproteases. PCSK9 is translated as a 692-amino acid proprotein that includes several domains found in other proprotein convertases, including an N-terminal signal sequence, a prodomain, a catalytic domain, and a cysteine-rich C-terminal domain (1-3). The PCSK9 catalytic domain shares high sequence similarity with the proteinase K family of subtilases and contains a catalytic triad (Asp 186 , His 226 , and Ser 386 ) responsible for autoprocessing (1, 4). PCSK9 processing occurs in the secretory pathway, presumably in the endoplasmic reticulum, and results in proteolytic cleavage occurring after Gln 152 (FAQ2SIP). This cleavage generates a stable PCSK9 heterodimer composed of a 14-kDa prodomain fragment and a mature 57-kDa fragment containing the catalytic and C-terminal domains (4, 5). Following processing, the PCSK9 heterodimer exits the ER and is eventually secreted (1). The prodomain of PCSK9 remains strongly bound to the mature protein after secretion, presumably inhibiting the catalytic activity of PCSK9 (1, 5, 6). To date, there is no conclusive evidence that the processed secreted form of PCSK9 can cleave any substrates in a catalytic serine-dependent manner.The first evidence that PCSK9 plays a significant role in regulating plasma low density lipoprotein (LDL) 3 cholesterol (LDL-C) levels was the identification of several missense mutations in PCS...

The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and b-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.

In addition to tRNA and 5S RNA, Escherichia coli contains several other small, stable RNA species; these are M1, 10Sa, 6S, and 4.5S RNA. Although these RNAs are initially synthesized as precursor molecules, relatively little is known about their maturation. The data presented here show that 3 exoribonucleolytic trimming is required for the final maturation of each of these molecules. As found previously with tRNA, but not 5S RNA, any one of a number of exoribonucleases can carry out the trimming reaction in vivo, although RNases T and PH are most effective. In their absence, large amounts of immature molecules accumulate for most of the RNAs, and these can be converted to the mature forms in vitro by the purified RNases. A model is proposed that identifies a structural feature present in all the small, stable RNAs of E. coli, and describes how this structure together with the RNases inf luences the common mechanism for 3 maturation.RNA molecules generally are synthesized as longer precursors that must undergo a series of processing reactions to remove extra residues and generate the mature, functional forms (1). While information about these RNA processing reactions and the RNases that catalyze them is accumulating, there still are major gaps in our knowledge of these pathways, particularly with regard to the less abundant classes of RNA.In Escherichia coli, in addition to tRNA and 5S RNA, four other small, stable RNA species are known. These include M1 RNA (377 nucleotides), the catalytic subunit of RNase P (2); 10Sa or tmRNA (363 nucleotides), which is involved in tagging prematurely terminated polypeptides for ultimate degradation (3); 4.5S RNA (114 nucleotides), a component of the prokaryotic signal recognition particle needed for protein secretion (reviewed in ref. 4); and 6S RNA (181-184 nucleotides), part of an 11S ribonucleoprotein complex of unknown function (5, 6). Each of these RNAs is initially synthesized with additional residues at its 5Ј and 3Ј ends that are removed during the course of maturation. The endoribonucleases, RNase P, RNase III, and RNase E, are thought to be involved in generating the mature 5Ј termini of some of these molecules, or in some instances, in removing portions of the 3Ј trailer sequence (7-10). However, no information is available on how the mature 3Ј termini are made.In previous studies, we showed that the 3Ј termini of tRNA and 5S RNA are generated by exoribonucleolytic trimming reactions (11)(12)(13)(14). In the case of tRNA, multiple exoribonucleases can participate in the 3Ј maturation process, with RNase PH and RNase T providing most of the processing activity (11)(12)(13)15). In contrast, 3Ј maturation of 5S RNA is completely dependent on RNase T (14). In this paper we demonstrate that exoribonucleolytic trimming reactions also are responsible for generating the mature 3Ј termini of the other small, stable RNAs, and we define which RNases participate in the process. A general model for 3Ј maturation of stable RNAs is also presented.

Polyadenylation at the 3 terminus has long been considered a specific feature of mRNA and a few other unstable RNA species. Here we show that stable RNAs in Escherichia coli can be polyadenylated as well. RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length. The polyadenylation process depends on the presence of poly(A) polymerase I. A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs. These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism.Cellular RNA molecules have long been divided into two groups. The first group, the stable RNAs, includes mainly rRNA, tRNA, and a variety of other small RNAs, and represents Ͼ95% of the total cellular RNA population. These RNAs have long lifetimes relative to the generation times of the cells in which they reside. The second group, unstable RNAs, consists primarily of mRNAs, which are a small fraction of the total RNA population and have half-lives that are usually much shorter than the generation time of the cell. The unstable RNAs generally contain a poly(A) tract at their 3Ј ends that contributes to their turnover; for mRNAs, poly(A) contributes to their role in translation (1-5). However, polyadenylation of stable RNAs has rarely been seen, and has not been considered important for stable RNA metabolism.In previous studies of the maturation of tRNA (6, 7), 5S RNA (8), and other small, stable RNAs (4.5S, 6S, M1, and tmRNA) (9) in Escherichia coli, it was shown that exoribonucleolytic trimming was a necessary step in the formation of the 3Ј termini of these various molecules. Thus, in the absence of the requisite 3Ј to 5Ј exoribonucleases, precursor products with extra nucleotides at their 3Ј ends accumulated. In some instances, however, these extra sequences were longer than expected. For example, 5S RNA is known to be released from long rRNA transcripts by RNase E endonucleolytic cleavages that leave three additional residues at each end of the processing intermediate (10-13). However, products were found to accumulate in some exoribonuclease-deficient strains that contained as many as 10 extra nucleotides at their 3Ј ends (8). Likewise, in a recent study of the maturation of M1 RNA, the catalytic subunit of RNase P, products with up to six additional 3Ј residues were observed (9), despite the fact that RNase E is thought to cleave this precursor at a position only one or two nucleotides downstream from the mature 3Ј terminus (14,15).In this paper we provide an explanation for these unexpected observations. To do so, we determined the nucleotide sequences at the 3Ј ends of the 5S and M1 RNA products that accumulate in the multiple exoribonuclease-deficient cells. This was acco...

Ribosomal RNAs are generally synthesized as long, primary transcripts that must be extensively processed to generate the mature, functional species. In Escherichia coli, it is known that the initial 30S precursor is cleaved during its synthesis by the endonuclease RNase III to generate precursors to the 16S, 23S, and 5S rRNAs. However, despite extensive study, the processes by which these intermediate products are converted to their mature forms are poorly understood. In this article, we describe the maturation of 23S rRNA. Based on Northern analysis of RNA isolated from a variety of mutant strains lacking one or multiple ribonucleases, we show that maturation of the 39 terminus requires the action of RNase T, an enzyme previously implicated in the end turnover of tRNA and in the maturation of small, stable RNAs. Although other exoribonucleases can participate in shortening the 39 end of the initial RNase III cleavage product, RNase T is required for removal of the last few residues. In the absence of RNase T, 23S rRNA products with extra 39 residues accumulate and are incorporated into ribosomes, with only small effects on cell growth. Purified RNase T accurately and efficiently converts these immature ribosomes to their mature forms in vitro, whereas free RNA is processed relatively poorly. In vivo, the processing defect at the 39 end has no effect on 59 maturation, indicating that the latter process proceeds independently. We also find that a portion of the 23S rRNA that accumulates in many RNase T -cells becomes polyadenylated because of the action of poly(A) polymerase I. The requirement for RNase T in 23S rRNA maturation is discussed in relation to a model in which only this enzyme, among the eight exoribonucleases present in E. coli, is able to efficiently remove nucleotides close to the double-stranded stem generated by the pairing of the 59 and 39 termini of most stable RNAs.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) has recently emerged as a major regulator of plasma low-density lipoprotein cholesterol (LDLc) and is a promising therapeutic target for treating coronary heart Press, October 19, 2010 DOI 10.1194 Abbreviations: AF, Alexa Fluor; CETP, cholesteryl ester transfer protein; CHD, coronary heart disease; DELFIA, dissociation-enhanced lanthanide fl uorescence immunoassays; HDLc, high-density lipoprotein cholesterol; LDLc, low-density lipoprotein cholesterol; LDLr, lowdensity lipoprotein receptor; MAb, monoclonal antibody; PEG, polyethylene glycol 6000; TCEP, Tris(2-carboxyethyl)phosphine; TG, triglycerides; TR-FRET, time-resolved fl uorescence resonance energy transfer. Published, JLR Papers in1 The atomic coordinates and structure factors have been deposited in the Protein Data Bank, accession code 2xtj.3 Current address: Okairos S.r.l.,