Degradosome

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The degradosome is a multiprotein complex present in most bacteria that is involved in the processing of ribosomal RNA and the degradation of messenger RNA and is regulated by Non-coding RNA. It contains the proteins RNA helicase B, RNase E and Polynucleotide phosphorylase. [1]

Contents

The store of cellular RNA in the cells is constantly fluctuating. For example, in Escherichia coli , Messenger RNA's life expectancy is between 2 and 25 minutes, in other bacteria it might last longer. Even in resting cells, RNA is degraded in a steady state, and the nucleotide products of this process are later reused for fresh rounds of nucleic acid synthesis. RNA turnover is very important for gene regulation and quality control.

All organisms have various tools for RNA degradation, for instance ribonucleases, helicases, 3'-end nucleotidyltransferases (which add tails to transcripts), 5'-end capping and decapping enzymes and assorted RNA-binding proteins that help to model RNA for presentation as substrate or for recognition. Frequently, these proteins associate into stable complexes in which their activities are coordinate or cooperative. Many of these RNA metabolism proteins are represented in the components of the multi-enzyme RNA degradosome of Escherichia coli, which is constituted by four basic components: the hydrolytic endo-ribonuclease RNase E, the phosphorolytic exo-ribonuclease PNPase, the ATP-dependent RNA helicase (RhIB) and a glycolytic enzyme enolase.

The RNA degradosome was discovered in two different laboratories while they were working on the purification and characterization of E. coli, RNase E and the factors that could have an influence on the activity of the RNA-degrading enzymes, concretely, PNPase. It was found while two of its major compounds were being studied.

Structure

The composition of this multienzyme may vary depending on the organism. The multiprotein complex RNA degradosome in E. coli consists of 4 canonical components:

There are some alternate forms of the RNA degradosome with different proteins that have been reported. Supplementary alternate degradosome components are PcnB (poly A polymerase) and the RNA helicases RhlE and SrmB. Other alternate components during cold shock include RNA helicase CsdA. Additional alternate degradosome components during stationary phase include Rnr (RNase R) and the putative RNA helicase HrpA. Ppk (polyphosphate kinase) is another constituent that has been reported to be part of the complex, the same as RNA chaperone Hfq, PAP (prostatic acid phosphatase), other kinds of chaperones and ribosomal proteins. These have been found in cell-extracted degradosome preparations from E. coli . [4]

This would represent the basic structure of RNA Degradosome. The structure has been drawn symmetrically, however, it's a dynamic structure so the noncatalytic region of RNase E would form a random coil, and each of these coils would act independently from the other ones. Degradosome.jpg
This would represent the basic structure of RNA Degradosome. The structure has been drawn symmetrically, however, it's a dynamic structure so the noncatalytic region of RNase E would form a random coil, and each of these coils would act independently from the other ones.

The structure of RNA degradosome is not as rigid as it seems to be in the picture because this one is only a model to understand how it works. The RNA degradosome's structure is dynamic and each component interacts with the components that are close to it. So the structure is like a molecular domain where RNA can interact as a substrate with each of the components and when this happens, it is really difficult for RNA to scape from the complex. [3]

Functions

The RNA degradosome is a huge multi-enzyme association that is involved in RNA metabolism and post-transcriptional control of gene expression in numerous bacteria such as Escherichia coli and Pseudoalteromonas haloplanktis . The multi-protein complex also serves as a machine for processing structured RNA precursors in the course of their maturation. [5] [6]

RNA helicase is considered to help in the process of degradation to develop the double helix structure in RNA stem-loops. Occasionally, copurification of rRNA with degradosome is appreciated, which suggests that the complex may take part in rRNA and mRNA degradation. There is very little clear information about the role of degradosome. Looking into the steps of the degradation of a transcript in E. coli, what is known is that in the first place the endoribonucleases can cleave the substrates so that later the exoribonucleases can work on the products. RhIB has very little activity by itself but the interaction with RNAse E can stimulate it. [7] The role of enolase in the degradation process of RNA is still not properly described, apparently it helps the complex to be more specific during the process of degradation. [8] [9]

One particularly intriguing aspect of the bacterial RNA degradosome is the presence of metabolic enzymes in many of the studied complexes. In addition to the enolase enzyme present in the E. coli degradosome, the metabolic enzymes aconitase and phosphofructokinase have been identified in the C. crescentus and B. subtilis degradosomes respectively. [10] [11] The reason for the presence of these enzymes is currently unclear.

Degradosome activation

This multi-protein complex is stimulated by a non-coding RNA, called miRNA in Eukaryotic cells and sRNA in bacteria. Small sequences of aminoacid are usually used to target mRNA for its destruction. From here, there are two ways to do it: targeting translation-initiation region (TIR) or coding DNA sequence (CDS). Firstly, to attach sRNA to targeted mRNA a Hfq (chaperone protein) is needed. Once the attachment is done, if the complex Hfq-sRna ends on TIR, it blocks ribosome binding site (RBS) so ribosomes can not translate, and activates nucleases (RNase E) to eliminate mRNA. Another possibility is ending on another region, that makes the complex work as a finisher point of the translation. This way, the ribosomes can do their job of decoding, process that stops when they arrive to the complex, where all the destruction procedure is switched on. [5]

This picture shows RNA's degradation process with the specific phases. RNA's Degradation Process.png
This picture shows RNA's degradation process with the specific phases.

RNA degradation

The RNA's destruction process is very complicated. To make it easier to understand, we use as an example the mRNA degradation procedure in Escherichia coli because it is the best known process. It is mediated mainly by endo- and ribo- nucleases. The enzymes RNase II and PNPase (polynucleotide phosphorylase) degrade mRNA in a 3'→5' way. The degradosome has 4 compartments that have several ribonucleases. Initially, the synthesized RNA is a polyphosphate structure. This is why the dephosphorylation is needed, in order to obtain monophosphate by the action of a RNA pyrophosphohydrolase PppH. The transcripts have two parts: the phosphate terminal (P-terminal) and a stem-loop structure as an end. The P-terminal is endoribonucleolytically cleavaged by RNase E, while the stem-loop is digested by RNA helicases. If there are any secondary structures, the performance of polymerase PAP is needed to simplify the reduction by exoribonucleases such as PNPase. Finally, the scraps are processed by oligoribonucleases.

The process is analogous in other species and only changes in the enzymatic machinery. For example, bacillus subtilis instead of using RNase E as the endo-ribonuclease, it uses RNase Y or RNase J or in the archaea is used an exosome (vesicle) to this job. [5]

Evolution

The degradosome, which is dynamic in conformation, variable in composition and non-essential under determined laboratory conditions, has nevertheless been maintained throughout the evolution of many bacterial species (Archaea, Eukaryote, Escherichia coli, Mitochondria, etc.), due most likely to its diverse contributions in global cellular regulation. It has been experimentally demonstrated that the presence of degradosome is a selective benefit for E. coli. [5]

Degradosome-like structures have been thought to be part of many γ-proteobactria and have actually been found in other remote bacterial lineages. They are built upon RNase E. However, the composition of these degradosome-like assemblies is not always the same, it may be different on some proteic components.

The RNA degradosome of E. coli

Humans and other animals have E. coli as a commensal in their intestinal tract. It is one of the most studied organisms at laboratories and it has been a useful model for understanding genetic regulation in bacteria and other domains of life. The RNA degradosome of E. coli is a structure that plays diverse roles in RNA metabolism. It shares homologous components and functional analogy with similar assemblies found in all domains of life. One of its components is an ATP-dependent motor that is activated through protein-protein interactions and cooperates with the ribonucleases in an energy-dependent mode of RNA degradation. [5]

E. coli does not have a 5'→3' degradation pathway. Its mRNA does not have 5' capped ends and there are not any 5'→3' exonucleases known. The same thing happens to other eubacteria, hence the 5'→3' degradation pathway could be an exclusive trait of eukaryotic cells. [7]

See also

Related Research Articles

Ribosome Intracellular organelle consisting of RNA and protein functioning to synthesize proteins

Ribosomes ( ), also called Palade granules, are macromolecular machines, found within all cells, that perform biological protein synthesis. Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to form polypeptide chains. Ribosomes consist of two major components: the small and large ribosomal subunits. Each subunit consists of one or more ribosomal RNA (rRNA) molecules and many ribosomal proteins. The ribosomes and associated molecules are also known as the translational apparatus.

Ribonuclease Class of enzyme that catalyzes the degradation of RNA

Ribonuclease is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 and 3.1 classes of enzymes.

Ribonuclease H

Ribonuclease H is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/DNA substrate via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from bacteria to archaea to eukaryotes.

Ribonuclease P

Ribonuclease P is a type of ribonuclease which cleaves RNA. RNase P is unique from other RNases in that it is a ribozyme – a ribonucleic acid that acts as a catalyst in the same way that a protein-based enzyme would. Its function is to cleave off an extra, or precursor, sequence of RNA on tRNA molecules. Further, RNase P is one of two known multiple turnover ribozymes in nature, the discovery of which earned Sidney Altman and Thomas Cech the Nobel Prize in Chemistry in 1989: in the 1970s, Altman discovered the existence of precursor tRNA with flanking sequences and was the first to characterize RNase P and its activity in processing of the 5' leader sequence of precursor tRNA. Recent findings also reveal that RNase P has a new function. It has been shown that human nuclear RNase P is required for the normal and efficient transcription of various small noncoding RNAs, such as tRNA, 5S rRNA, SRP RNA and U6 snRNA genes, which are transcribed by RNA polymerase III, one of three major nuclear RNA polymerases in human cells.

Ribonuclease III

Ribonuclease III (BRENDA 3.1.26.3) is a type of ribonuclease that recognizes dsRNA and cleaves it at specific targeted locations to transform them into mature RNAs. These enzymes are a group of endoribonucleases that are characterized by their ribonuclease domain, which is labelled the RNase III domain. They are ubiquitous compounds in the cell and play a major role in pathways such as RNA precursor synthesis, RNA Silencing, and the pnp autoregulatory mechanism.

LSm

In molecular biology, LSm proteins are a family of RNA-binding proteins found in virtually every cellular organism. LSm is a contraction of 'like Sm', because the first identified members of the LSm protein family were the Sm proteins. LSm proteins are defined by a characteristic three-dimensional structure and their assembly into rings of six or seven individual LSm protein molecules, and play a large number of various roles in mRNA processing and regulation.

Exosome complex Protein complex that degrades RNA

The exosome complex is a multi-protein intracellular complex capable of degrading various types of RNA molecules. Exosome complexes are found in both eukaryotic cells and archaea, while in bacteria a simpler complex called the degradosome carries out similar functions.

Polynucleotide phosphorylase Class of enzymes

Polynucleotide Phosphorylase (PNPase) is a bifunctional enzyme with a phosphorolytic 3' to 5' exoribonuclease activity and a 3'-terminal oligonucleotide polymerase activity. That is, it dismantles the RNA chain starting at the 3' end and working toward the 5' end. It also synthesizes long, highly heteropolymeric tails in vivo. It accounts for all of the observed residual polyadenylation in strains of Escherichia coli missing the normal polyadenylation enzyme. Discovered by Marianne Grunberg-Manago working in Severo Ochoa's lab in 1955, the RNA-polymerization activity of PNPase was initially believed to be responsible for DNA-dependent synthesis of messenger RNA, a notion that got disproved by the late 1950s.

GcvB RNA

The gcvB RNA gene encodes a small non-coding RNA involved in the regulation of a number of amino acid transport systems as well as amino acid biosynthetic genes. The GcvB gene is found in enteric bacteria such as Escherichia coli. GcvB regulates genes by acting as an antisense binding partner of the mRNAs for each regulated gene. This binding is dependent on binding to a protein called Hfq. Transcription of the GcvB RNA is activated by the adjacent GcvA gene and repressed by the GcvR gene. A deletion of GcvB RNA from Y. pestis changed colony shape as well as reducing growth. It has been shown by gene deletion that GcvB is a regulator of acid resistance in E. coli. GcvB enhances the ability of the bacterium to survive low pH by upregulating the levels of the alternate sigma factor RpoS. A polymeric form of GcvB has recently been identified. Interaction of GcvB with small RNA SroC triggers the degradation of GcvB by RNase E, lifting the GcvB-mediated mRNA repression of its target genes.

RNase E 5′ UTR element

In molecular biology, the RNase E 5′ UTR element is a cis-acting element located in the 5′ UTR of ribonuclease (RNase) E messenger RNA (mRNA).

SroC RNA

The bacterial SroC RNA is a non-coding RNA gene of around 160 nucleotides in length. SroC is found in several enterobacterial species. This RNA interacts with the Hfq protein.

Hfq protein

The Hfq protein encoded by the hfq gene was discovered in 1968 as an Escherichia coli host factor that was essential for replication of the bacteriophage Qβ. It is now clear that Hfq is an abundant bacterial RNA binding protein which has many important physiological roles that are usually mediated by interacting with Hfq binding sRNA.

Pancreatic ribonuclease family

Pancreatic ribonuclease family is a superfamily of pyrimidine-specific endonucleases found in high quantity in the pancreas of certain mammals and of some reptiles.

Exoribonuclease II is an enzyme. This enzyme catalyses the following chemical reaction

RNase R, or Ribonuclease R, is a 3'-->5' exoribonuclease, which belongs to the RNase II superfamily, a group of enzymes that hydrolyze RNA in the 3' - 5' direction. RNase R has been shown to be involved in selective mRNA degradation, particularly of non stop mRNAs in bacteria. RNase R has homologues in many other organisms.

Non-stop decay

Non-stop decay (NSD) is a cellular mechanism of mRNA surveillance to detect mRNA molecules lacking a stop codon and prevent these mRNAs from translation. The non-stop decay pathway releases ribosomes that have reached the far 3' end of an mRNA and guides the mRNA to the exosome complex, or to RNase R in bacteria for selective degradation. In contrast to Nonsense-mediated decay (NMD), polypeptides do not release from the ribosome, and thus, NSD seems to involve mRNA decay factors distinct from NMD.

MicX sRNA

MicX sRNA is a small non-coding RNA found in Vibrio cholerae. It was given the name MicX as it has a similar function to MicA, MicC and MicF in E. coli. MicX sRNA negatively regulates an outer membrane protein and also a component of an ABC transporter. These interactions were predicted and then confirmed using a DNA microarray.

Ribonuclease E is a bacterial ribonuclease that participates in the processing of ribosomal RNA and the chemical degradation of bulk cellular RNA.

Retroviral ribonuclease H

The retroviral ribonuclease H is a catalytic domain of the retroviral reverse transcriptase (RT) enzyme. The RT enzyme is used to generate complementary DNA (cDNA) from the retroviral RNA genome. This process is called reverse transcription. To complete this complex process, the retroviral RT enzymes need to adopt a multifunctional nature. They therefore possess 3 of the following biochemical activities: RNA-dependent DNA polymerase, ribonuclease H, and DNA-dependent DNA polymerase activities. Like all RNase H enzymes, the retroviral RNase H domain cleaves DNA/RNA duplexes and will not degrade DNA or unhybridized RNA.

Ribonuclease T

Ribonuclease T is a ribonuclease enzyme involved in the maturation of transfer RNA and ribosomal RNA in bacteria, as well as in DNA repair pathways. It is a member of the DnaQ family of exonucleases and non-processively acts on the 3' end of single-stranded nucleic acids. RNase T is capable of cleaving both DNA and RNA, with extreme sequence specificity discriminating against cytosine at the 3' end of the substrate.

References

  1. ↑ Carpousis AJ (April 2002). "The Escherichia coli RNA degradosome: structure, function and relationship in other ribonucleolytic multienzyme complexes". Biochemical Society Transactions. 30 (2): 150–5. doi:10.1042/BST0300150. PMID   12035760.
  2. ↑ Bandyra KJ, Bouvier M, Carpousis AJ, Luisi BF (June 2013). "The social fabric of the RNA degradosome". Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1829 (6–7): 514–22. doi:10.1016/j.bbagrm.2013.02.011. PMC   3991390 . PMID   23459248.
  3. 1 2 Carpousis AJ (2007-09-26). "The RNA degradosome of Escherichia coli: an mRNA-degrading machine assembled on RNase E". Annual Review of Microbiology. 61 (1): 71–87. doi:10.1146/annurev.micro.61.080706.093440. PMID   17447862.
  4. ↑ EcoGene. "EcoGene". www.ecogene.org. Archived from the original on 2016-10-20. Retrieved 2016-10-19.
  5. 1 2 3 4 5 Górna MW, Carpousis AJ, Luisi BF (May 2012). "From conformational chaos to robust regulation: the structure and function of the multi-enzyme RNA degradosome". Quarterly Reviews of Biophysics. 45 (2): 105–45. doi:10.1017/S003358351100014X. PMID   22169164. S2CID   25761069.
  6. ↑ Aït-Bara S, Carpousis AJ (October 2010). "Characterization of the RNA degradosome of Pseudoalteromonas haloplanktis: conservation of the RNase E-RhlB interaction in the gammaproteobacteria". Journal of Bacteriology. 192 (20): 5413–23. doi:10.1128/JB.00592-10. PMC   2950506 . PMID   20729366.
  7. 1 2 Carpousis, A. J. (2002). "The Escherichia coli RNA degradosome: structure, function and relationship to other ribonucleolytic multienyzme complexes". Biochemical Society Transactions. 30 (2): 150–155. doi:10.1042/0300-5127:0300150.
  8. ↑ Brown T (2008-06-30). Genomas/ Genome (in Spanish). Ed. Médica Panamericana. ISBN   9789500614481.
  9. ↑ Garcia-Mena J. "Polinucleótido fosforilasa: una joya de las ribonucleasas". ResearchGate. Retrieved 18 October 2016.
  10. ↑ Hardwick SW, Chan VS, Broadhurst RW, Luisi BF (March 2011). "An RNA degradosome assembly in Caulobacter crescentus". Nucleic Acids Research. 39 (4): 1449–59. doi:10.1093/nar/gkq928. PMC   3045602 . PMID   20952404.
  11. ↑ Cho KH (2017). "The Structure and Function of the Gram-Positive Bacterial RNA Degradosome". Frontiers in Microbiology. 8: 154. doi: 10.3389/fmicb.2017.00154 . PMC   5289998 . PMID   28217125.
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