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Rabbit Reticulocyte Lysate Translation Systems: Tools for the analysis of translational regulation

Posted on by Gary Kobs
TEM of Norovirus particles. Photo Credit: Charles D. Humphrey, Centers for Disease Control and Prevention
TEM of Norovirus particles. Photo Credit: Charles D. Humphrey, Centers for Disease Control and Prevention

Rabbit Reticulocyte Lysate Translation Systems are used in the identification of mRNA species, the characterization of their protein products and the investigation of transcriptional and translational control. Rabbit Reticulocyte Lysate is prepared from New Zealand white rabbits. After the reticulocytes are lysed, the extract is treated with micrococcal nuclease to destroy endogenous mRNA and thus reduce background translation to a minimum.

Untreated Lysate is prepared from New Zealand white rabbits in the same manner as treated lysates with the exception that it is not treated with micrococcal nuclease. Unlike a coupled system that initiates transcription/translation from DNA, the RNA-based rabbit reticulocyte can be used for the direct investigation of transcriptional/translational control and the replication of RNA-based viruses.


References
Characterization of translation regulation (i.e., UTRs, Capping, IRES)

  1. Nguyen, H-L .et al. (2013) Expression of a novel mRNA transcript for human microsomal epoxide hydrolase is regulated by short reading frames within it 5’ –untranslated region. RNA. 19, 752–66.
  2. Wei, J. et al. (2013) The stringency of start codon selection in the filamentous fungus Neurospora crass. J. Biol. Chem. 288, 9549–62.
  3. Paek Ki-Y. et al. (2012) Cap-Dependent translation without base-by-base scanning of an messenger ribonucleic acid. Nucl. Acid. Res. 40, 7541–51.
  4. Se, and NH. Su.W. et al. (2011) Translation, stability, and resistance to decapping of mRNA containing caps substituted in the triphosphate with BH3. RNA 17, 978–88.
  5. Anderson, D. et al. (2011) Nucleoside modifications in RNA limit activation of 2’-5’ oligoadenylate synthetase and increase resistance to cleavage by RNase L. Nucl. Acid. Res. 39, 9329-38.

RNA virus Characterization

  1. Vashist, S. et al. (2012) Identification of RNA-protein interaction networks involved in the Norovirus life cycle. J. Vir. 86, 11977–90.
  2. Soto-Rifo, R. et al. (2012) Different effects of the TAR structure on HIV-1 and HIV-2 genomics RNA translation. Nucl. Acids. Res. 40, 2653–67.
  3. Poyry, T. et al. (2011) Mechanisms governing the selection of translation initiation sites on Foot-and-Mouth Disease Virus RNA. J.Vir. 85, 10178–88.
  4. Cheng, E. et al. (2011) Characterization of the interaction between Hantavirus nucleopcapsid protein and ribosomal protein S19. J. Biol. Chem. 286, 11814–24.
  5. Vera-Otarola, J. et al. (2011) The Andes Hantavirus NSs Protein is expressed from the Viral mRMA by a leaky scanning mechanism. J. Vir. 86, 2176–87.

Characterization of Ubiquitination Using Cell-Free Expression

Posted on by Gary Kobs

Ubiquitination refers to the post translational modification of a protein by attachment of one or more ubiquitin monomers. The most prominent function of ubiqutin is labeling proteins for proteasome degradation. In addition to this function ubiquitination also controls the stability, function and intracellular localization of a wide variety of proteins.

Cell free expression can be used to characterize ubiquitation of proteins. Target proteins are expressed in a rabbit reticulocyte cell free system (supplemented with E1 ubiquitin activating enzyme, E2 ubiquitin –conjugating enzyme, and ubiquitin). Proteins that have been modified can be analyzed by a shift in migration on polyacrylamide gels.

The following references illustrate the use of cell free expression for this application.

Jung, Y.S. et al. (2011) The p73 Tumor Suppressor Is Targeted by Pirh2 RING Finger E3 Ubiquitin Ligase for the Proteasome-dependent Degradation. J. Biol. Chem. 286, 35388–95.

Su, C-H, et al. (2010) 14-3-3sigma exerts tumor-suppressor activity mediated by regulation of COP1 stability. Cancer. Res. 71, 884–94.

Naoe, H. et al. (2010). The anaphase-promoting complex/cyclosome activator Cdh1 modulates Rho GTPase by targeting p190 RhoGAP for degradation. Mol. Cell. Biol. 30, 3994-05.

de Thonel, A. et al. (2010) HSP27 controls GATA-1 protein level during erythroid cell differentiation. Blood 116, 85–96.

Kaneko, M. et al. (2010) Loss of HRD1-mediated protein degradation causes amyloid precursor protein accumulation and amyloid-beta generation. J. Neurosci. 30, 3924–32.

Optimized Protein Expression: Flexi Rabbit Reticulocyte Lysate

Posted on by Gary Kobs
A protein chain being produced from a ribosome.

mRNAs commonly exhibit differing salt requirements for optimal translation. Small variations in salt concentration can lead to dramatic differences in translation efficiency. The Flexi® Rabbit Reticulocyte Lysate System allows translation reactions to be optimized for a wide range of parameters, including
Mg2+ and K+ concentrations and the choice of adding DTT. To help optimize Mg2+ for a specific message, the endogenous Mg2+ concentration of each lysate batch is stated in the product information included with this product.

The following references utilize the features of Flexi Rabbit Reticulocyte Lysate System to investigate certain parameters of translation:

Vallejos, M. et al. (2010)The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation. Nucl Acids Res. 38, 618–32. Identification of internal ribosomal ribosomal entry site in the 5’ untranslated region of the mouse mammary tumor virus mRNA.

Spriggs, K. et al. (2009) The human insulin receptor mRNA contains a functional internal ribosome entry segment. Nucl. Acids. Res. 17, 5881–93. Identification of a functional internal ribosome entry site in the human insulin receptor mRNA.

Powell, M. et al. (2008) Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein. RNA 14, 2394–06. Analysis of the mRNA signals involved in the expression of influenza B virus BM2 protein.

Sato, V. et al. (2007) Measles virus N protein inhibits host translation by binding to eIF3-p40. J. Vir. 81, 11569–76. Charaterized the effect of the measles virus N protein binding to the translation initiation factor eIF3-p40 on the expression of various proteins in rabbit reticulocyte lysate.

Hirao, K. et al. (2006) EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J. Biol. Chem. 281, 9650–58. The EDEM3 protein was expressed in the presence of canine microsomal membranes to establish that co-translational translocation occurs into the endoplasmic reticulum.

Shenvi, C. et al. (2005) Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations–implications for the study of ribosome dynamics. RNA 11, 1898–08. Characterization of ribosome dynamics under different ionic conditions.

Nair, A. et al. (2005) Regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mRNA-binding protein and its endogenous association with luteinizing hormone receptor mRNA in the ovary. J. Biol. Chem. 280, 42809–16. Examined the affect of luteinizing hormone receptor mRNA binding protein on transltional suppression of luteinizing hormone receptor RNA.

Protease K Protection Assay: Cell Free Expression Application

Posted on by Gary Kobs

Microsomal vesicles are used to study cotranslational and initial posttranslational processing of proteins. Processing events such as signal peptide cleavage, membrane insertion, translocation and core glycosylation can be examined by the transcription/translation of the appropriate DNA in the TNT® Lysate Systems when used with microsomal membranes.

The most general assay for translocation makes use of the protection afforded the translocated domain by the lipid bilayer of the microsomal membrane. In this assay protein domains are judged to be translocated if they are observed to be protected from exogenously added protease. To confirm that protection is due to the lipid bilayer addition of 0.1% non-ionic detergent (such as Triton® X-100) solubilizes the membrane and restores susceptibility to the protease.

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Many proteases have proven useful for monitoring translocation in this fashion including Protease K or Trypsin.

The following are examples illustrating this application:

  1. Minn, I. et al. (2009) SUN-1 and ZYG-12, mediators of centrosome-nucleus attachment, are a functional SUN/KASH pair in Caenorhabditis elegans. Mol. Biol. Cell. 20, 4586–95.
  2. Padhan, K. et al. (2007) Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin. J.Gen.Virol. 88, 3067–77.
  3. Tews, B.A. et al. (2007) The pestivirus glycoprotein Erns is anchored in plane in the membrane via an amphipathic helix. J.Biol.Chem. 282, 32730–41.
  4. Pidasheva, S. et al. (2005) Impaired cotranslational processing of the calcium-sensing receptor due to signal peptide missense mutations in familial hypocalciuric hypercalcemia. Hum. Mol. Gen. 14, 1679–90.
  5. Smith, D. et al. (2002) Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation. J. Immun. 169, 99–107.