Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Summary

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

Abstract

The 3’ end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.

Processing of the 3’ end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long ³²P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5’ cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3’ end processing of HIV-1 mRNAs.

Introduction

The biosynthesis of most mature eukaryotic message RNAs (mRNAs) requires several post-transcriptional modifications such as capping, splicing and polyadenylation. These modifications are generally coupled to ensure correct processing¹ and strongly increase the stability of the mRNA.

The 3’ end formation of mammalian pre-mRNAs is generated by endonucleolytic cleavage of the nascent RNA followed by addition of adenylate residues to the 5’ cleaved product by poly(A) polymerase (PAP)^2-4. In mammals, cleavage is accomplished by a multicomponent protein complex, of approximately 800 kDa, which assembles on specif....

Protocol

1. Adaptation of Adherent Cells into Suspension

(This is an optional step. Suspension cells generally make better nuclear extracts, however cells grown in plates may also be used.)

1. Adapt adherent cells for growth in suspension using Joklik's modified MEM supplemented with 5-10% newborn calf serum or fetal bovine serum and 1% L-glutamine: penicillin-streptomycin. Propagate cells in spinner flasks with a filter cap at 37 °C with 8% CO₂.
2. When adapting cells, start with 100 ml in a 500 ml spinner flask. Maintain a minimum 0.3 x 10⁶ cells/ml and replace the medium every 1-2 days until the....

Results

Representative results of a cleavage assay of the RNA poly(A) sites of HIV-1 (Figure 2). We can observe the uncleaved RNA substrate, which is the slowest migrating band at the top of the gel. The specific cleaved product is the most intense shorter fragment band that runs faster in the gel at the expected size, and is specifically absent from the cleavage assay of the mutpoly(A) control that contains a point mutation in the polyA sequence (mutPolyA) RNA substrate. Degradation products of the input substr.......

Discussion

The in vitro pre-mRNA 3’ cleavage reaction, carried out in HeLa cell nuclear extracts or with cleavage factors fractionated from these extracts, has enabled identification of the core cleavage factors and their main complexes^16-21. Many more proteins associated with these factors have recently been identified²², and the in vitro reaction may continue to shed light on how these proteins contribute to the reaction. Perhaps because the reaction in vivo appears to be cot.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 L Celstir flask	Wheaton	356884	Different sizes available
4 position slow speed stirrer	VWR	12621-076
Swinging bucket centrifuge	Beckman Coulter		Allegra x-15R with SX4500 Rotor
Ultracentrifuge	Beckman Coulter		Optima L-100 XP Ultra with SW41 Rotor
Table top centrifuge 5417R	Eppendorf		Refridgerated
Thermomixer incubator	Eppendorf
250 ml conical tubes	Corning	430776
50 ml conical tubes	BD Falcon	352098
Ultraclear centrifuge tubes	Beckman Coulter	344059
JOKLIK modified MEM	Lonza	04-719Q
Fetal bovine serum	Atlas	F-0500-A	Heat inactivated
L-glut:pen:strep	Gemini Bio-Products	400-110
1 M Tris-HCl pH 8.0	Mediatech	46-031-CM
Magnesium chloride	Fisher	BP214-500
Potassium chloride	MP Biomedicals	194844
HEPES	Fisher	BP310-500
DTT	Alexis Biomedicals	280-001-G-010
Glycerol	Fisher	BP229-4
5 M sodium chloride solution	Mediatech	46-032-CV
EDTA 0.5 M solution	Sigma-Aldrich	E7889-100ml
EDTA	Fisher	BP120-500
PMSF	Thermo Scientific	36978
Ammonium sulfate	Fisher	A702-500
Complete EDTA-free protease inhibitor cocktail tablets	Roche	04 693 132 001
Slide-A-Lyzer Dialysis Cassettes Kit	Thermo Scientific	66372	MWCO 7,000 0.5 ml-3 ml
15 ml Dounce tissue grinder set	Sigma-Aldrich	D9938-1SET	Different sizes available
Expand high fidelity PCR kit	Roche	11 732 650 001
10 mM dNTP Mix	Invitrogen	Y02256	10 mM each nucleotide
MaxiScript SP6/T7 kit	Ambion	AM1322
m7G(5')ppp(5') G RNA cap	New England Biolabs	S1404S
Century Marker Template Plus	Ambion	AM7782
Easytides UTP [α-32P] 250 μCi	Perkin Elmer	BLU507H250UC
Gel loading buffer II	Ambion	8546G
DEPC treated water	Ambion	AM9906
10x TAE	Fisher	BP13354
10x TBE	Ameresco Life Sciences	0658-4L
10x PBS	Fisher	BP399-20
Urea	Fisher	BP169-212
Ammonium persulfate	Bio-Rad	161-0700
TEMED	Fisher	BP150-20
Ammonium acetate	Fisher	A637-500
40% 19:1 acrylamide:bis-acrylamide	Bio-Rad	161-0144
Glycogen	Roche	10 901 393 001
100% absolute ethanol 200 proof	Acros	61509-0040
Acid phenol:chloroform	Ambion	9720	For RNA
Scintilation fluid	Fisher	SX18-4
Rnase inhibitor	Promega	N261B
Polyvinyl alcohol- PVA	Sigma-Aldrich	P8136-250G
Creatine phosphate	Calbiochem	2380
100 mM dATP	Fisher	BP2560-4
SDS	Acros	23042-5000
Proteinase K	Fisher	BP1700-100
Adjustable sequencing unit	Sigma-Aldrich	Z351881-1EA
Binder clips	Office Depot	838-056
20 cm x 42 cm glass plates	Sigma-Aldrich	Z352543	1 SET
20 cm x 22 cm glass plates	Sigma-Aldrich	Z35252-7	1 SET
20 cm x 42 cm aluminum cooling plates	Sigma-Aldrich	Z352667	1 EA
0.4 mm x 22 cm spacers	Sigma-Aldrich	Z35230-6	1 SET
0.4 mm x 42 cm spacers	Sigma-Aldrich	Z352314-1	1 SET
8-well comb	Sigma-Aldrich	Z35195-4	1 EA
16-well comb	Sigma-Aldrich	Z351962	1 EA
32-well comb	Sigma-Aldrich	Z351970	1 EA
Gel repel coating	C.B.S. Scientific	SGR-0401	or SGR-0101 for individual bottle
Gel loading tips	Rainin	GT-10-4	0.1-10 μl
Sequencing PowerPac HV	Bio-Rad	PowerPac HV	5,000 V/500 mA/400 W
Gel Dryer Model 583	Bio-Rad	Model 583
Hydrotech vacuum pump for gel dryer	Bio-Rad
Glogos II Glow-in-the-dark markers	Agilent	420201
Film 8 x 10	Midsci	BX810
Film 14 x 17	Phenix	F-BX1417
Autoradiography cassette	Fisher	FBCA 1417	8 x 10 size available