Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Summary

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Abstract

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Introduction

Regulation of gene expression in cells is controlled by transcriptional, posttranscriptional and posttranslational mechanisms. Advances in deep RNA sequencing allow the study of steady-state mRNA levels on a genome-wide scale at an unprecedented level. However, recent findings revealed that steady-state mRNA level does not always correlate with protein production^1,2. The fate of an individual transcript is very complex and depends on many factors like internal/external stimuli, stress, etc. Regulation of gene expression during protein synthesis provides another layer of expression control necessary fo....

Protocol

All animal treatments and handling of tissues obtained in the study were performed according to protocols approved by the Institutional Animal Care and Use Committee at the Texas Tech University Health Science Center in accordance with the National Institutes of Health animal welfare guidelines, protocol number 96005. Please sacrifice vertebrate animals and prepare tissues according to the guidelines from the Institutional Animal Care and Use Committee. If lacking such a committee, please refer to the National Institutes of Health animal welfare guidelines. Adult (>60 day old) C57BL/6 mice were used. All animals and tissues were obtained according to protocols app....

Results

In this study, we describe the application of the polysomal profiling technique to three different sources: parasitic Leishmania major, cultured human cells, and mouse testis. Leishmania cells freely grow in the liquid media in suspension, cultured human cells grow in the adherent monolayer on plates, and the mouse testis represents a tissue sample. The method can be easily adjusted to other types of freely grown cells in suspension, different types of tissues, or from a.......

Discussion

Polysome fractionation by sucrose gradient combined with RNA and protein analysis of fractions is a powerful method to analyze translational status of individual mRNAs or the whole translatome as well as roles of protein factors regulating translational machinery during normal physiological or disease state. Polysomal profiling is an especially suitable technique to study translational regulation in organisms such as trypanosomatids including Leishmania where transcriptional control is largely absent and gene ex.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Instruments:
Gradient master	Biocomp Instruments Inc.	108
Piston Gradient Fractionator	Biocomp Instruments Inc.	152
Fraction collector	Gilson, Inc.	FC203B
NanoDrop One	Thermo Scientific	NanoDrop One
Nikon inverted microscope	Nikon	ECLIPSE Ts2-FL/Ts2
2720 Thermal Cycler	Applied Biosystems by Life Technologies	4359659
CO2 incubator	Panasonic Healthcare Co.	MCO-170A1CUV
HERATHERM incubator	Thermo Scientific	51028063
Biological Safety Cabinet, class II, type A2	NuAire Inc.	NU-543-400
Revco freezer	Revco Technologies	ULT1386-5-D35
Beckman L8-M Ultracentifuge	Beckman Coulter	L8M-70
Centrifuge	Eppendorf	5810R
Centrifuge	Eppendorf	5424
Ultracentrifuge Rotor SW41	Beckman Coulter	331362
Swing-bucket rotor	Eppendorf	A-4-62
Fixed angle rotor	Eppendorf	F-45-30-11
Quant Studio 12K Flex Real-Time PCR machine 285880228	Applied Biosystems by life technologies	4470661
TC20 Automated cell counter	Bio-Rad	145-0102
Hemacytometer	Hausser Scientific	02-671-51B
Software
Triax software	Biocomp Instruments Inc.
Materials:
Counting slides, dual chamber for cell counter	Bio-Rad	145-0011
1.5 mL microcentrifuge tube	USA Scientific	1615-5500
Open-top polyclear centrifuge tubes, (14 mm x 89 mm)	Seton Scientific	7030
Syringe, 5 mL	BD	309646
BD Syringe 3 mL23 Gauge 1 Inch Needle	BD	10020439
Nunclon Delta Surface plate, 14 cm	Thermo Scientific	168381
Nunclon Delta Surface plate, 9 cm	Thermo Scientific	172931
Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES	Thermo Scientific	569-0020
BioLite 75 cm3 flasks	Thermo Scientific	130193
Nunc 50 mL conical centrifuge tubes	Thermo Scientific	339653
Chemicals:
Trizol LS	Ambion by Life Technologies	10296028
HEPES	Fisher Scientific	BP310-500
Trizma base	Sigma	T1378-5KG
Dulbecco's Modified Eagle's Medium-high glucose (DMEM)	Sigma	D6429-500ML
Fetal Bovine Serum (FBS)	Sigma	F0926-50ML
Penicillin-Streptomycin (P/S)	Sigma	P0781-100ML
Lipofectamine 2000	Invitrogen	11668-019
Dulbecco's phosphate buffered saline (DPBS)	Sigma	D8537-500ML
Magnesium chloride hexahydrate (MgCl2x6H2O)	Acros Organics	AC413415000
Potassium Chloride (KCl)	Sigma	P9541-500G
Nonidet P 40 (NP-40)	Fluka (Sigma-Aldrich)	74385
Recombinant Rnasin Ribonuclease Inhibitor	Promega	N2511
Heparin sodium salt	Sigma	H3993-1MU
cOmplete Mini EDTA-free protease inhibitors	Roche Diagnostics	11836170001
Glycogen	Thermo Scientific	R0551
Water	Sigma	W4502-1L
Cycloheximide	Sigma	C7698-1G
Chloroform	Fisher Scientific	194002
Dithiotreitol (DTT)	Fisher Scientific	BP172-5
Ethidium Bromide	Fisher Scientific	BP-1302-10
Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)	Fisher Scientific	S316-212
Optimem	Life Technologies	22600050
Puromycin dihydrochloride	Sigma	P8833-100MG
Sucrose	Fisher Scientific	S5-3KG
Trypsin-EDTA solution	Sigma	T4049-100ML
Hgh Capacity cDNA Reverse Transcriptase Kit	Applied Biosystems by life technologies	4368814
Power SYBR Green PCR Master Mix	Applied Biosystems by life technologies	4367659
HCl	Fisher Scientific	A144SI-212
Isopropanol	Fisher Scientific	BP26324
Potassium Hydroxide (KOH)	Sigma	221473-500G
Anti-RPL11 antibody	Abcam	ab79352
Ribosomal protein S6 (C-8) antibody	Santa Cruz Biotechnology Inc.	sc-74459
1xM199	Sigma	M0393-10X1L
Lithium cloride	Sigma	L-9650
Dimethyl sulfoxide (DMSO)	Fisher Scientific	D128-500
Gel Loading Buffer II	Thermo Scientific	AM8546G
UltraPure Agarose	Thermo Scientific	16500-100
Trichloracetic acid (TCA)	Fisher Scientific	A322-100
SuperSignal West Pico PLUS chemiluminescent substrate	Thermo Scientific	34580
Formaldehyde	Fisher Scientific	BP531-500
Sodium Dodecyl Sulfate (SDS)	Sigma	L5750-1KG
Phenylmethylsulfonyl fluoride (PMSF)	Sigma	P7626-5G
RNeasy Mini kit	Qiagen	74104
Adenosine 5′-triphosphate disodium salt hydrate (ATP)	Sigma	A1852-1VL
Cytosine 5'-triphosphate disodium salt hydrate (CTP)	Sigma	C1506-250MG
Uridine 5'-triphosphate trisodium salt hydrate (UTP)	Sigma	U6625-100MG
Guanosine 5'-triphosphate sodium salt hydrate (GTP)	Sigma	G8877-250MG
SP6 RNA Polymerase	NEB	M0207S
Pyrophoshatase	Sigma	I1643-500UN
Spermidine	Sigma	S0266-1G