Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

ABSENCE OF DETECTABLE RNA LIGASE ACTIVITY Elliott

Bedows Department

RESEARCH COMMUNICATIONS

IN EUKARYOTIC CELLS

and Joseph T. Wachsman of Microbiology and

Richard I. Gumport of Biochemistry and School of Basic

Department

Medical

Sciences

University of Illinois Urbana, Illinois 61801

Received

October

14,1975

SUMMARY: Reports of the existence of eukaryotic RNA ligases may be incorrect. Evidence for this activity has been based upon the conversion of [5'-32~]terminated oligoribonucleotides to an alkaline phosphatase resistant form and upon the detection of radioactive ribonucleoside monophosphates after alkaline hydrolysis of the reaction products. Although we have in part confirmed these observations, we find the labeled ribonucleoside monophosphate to be the 5'-isomer, and not the expected 2'(3')-isomer. In addition, roughly equivalent amounts of ribonucleoside monophosphate were observed whether or not alkaline hydrolysis was performed. We conclude that the existence of RNA ligase activity in eukaryotic cells is suspect. INTRODUCTION:

RNA ligase

shown to catalyze bonds

with

formation

the

various of alkaline

The above

assays

activities

amounts

we feel

Using

of NMPs whether were

RNA ligase that

the

formation

used

these

activity

existence

hydrolysis

exclusively in

several

of RNA ligase

assayed

by measuring

ribonucleoside

for

monophos-

products.

possible

we have

eukaryotic

found

RNA

similar In addition,

was performed.

the 5'-isomers.

the

[5'-'LP]-terminated

of the reaction

procedures,

and has been

of phosphodiester

from

of labeled

in the search

or not alkaline almost

has been material

hydrolysis

have been

(2,5,7).

the NMFs detected to detect

alkaline

resistant

g. coli

formation

It

(l-7).

phosphatase

following

in T4-infected

and intermolecular

and by the

(NM%)

ligase

intra-

RNA substrates

oligoribonucleotides phates

was discovered

We have been unable

different

eukaryotic

activity

in eukaryotic

cell cells

lines, is

and in

question. of average chain length 65 (P-L Laboratories) MATERIALS AND METHODS: Poly(1) was converGta[5'-32P]-derivative as described previously (3). Reaction mixtures were incubated at 37O and were assayed for alkaline phosphatase (BAP C,

1100

BIOCHEMICAL

Vol. 67, No. 3, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Worthington) resistance, and/or NMP formation by adsorbing and eluting nucleotides from charcoal before and after alkaline hydrolysis (3). Nucleotides were separated by high voltage electrophoresis (8), eluted and chromatographed in a solution containing 6 parts [M ammonium acetate and 3.3 mM EDTA saturated with boric acid (adjusted to pH 7.0 with concentrated NH40H)] and 4 parts [absolute ethanol] (v/v) on cellulose coated thin layer plates (Eastman), to separate 2'(3')-NMPs from the 5'-isomers. RESULTS:

With

co-migrated sequently

eukaryotic

with

the

recovered

chromatography

(Table

by alkaline tion

amounts

I).

greater

marker

5'-IMP

during 2'(3')-IMP

hydrolysis

(Table

region

linkage

90% of the counts

the

2'(3')(data

detected This

at pH 4.0,

when the isomers

electrophoresis

I).

than

on electrophoresis

At pH 4.0 both

of

of a phosphodiester

extracts,

2'(3')-IMP in the

AMP and CMP co-migrate the small

cell

were

not

shown).

in some experiments finding

between

is

inconsistent

a [5'- 32 P]phosphate

were

separated

and 5'-isomers

which subby

of IMP, In addition,

were

not increased

with

the

forma-

and a 3'-hydroxyl

B 0.03

D.0

I

Figure 1. Effect of added L cell extract on T4 RNA ligase activity. T4 RNA Egase was added to a 50 ~1 reaction in the presence (B) and in the absence (0) of 0.4 mg/ml of L cell extract, or L cell extract was assayed alone (A). All were assayed as described in materials and methods, In A, 0.65 units of T4 RNA ligase were added to each reaction, and in B, 0.06 units of enzyme were added. The pmoles of [32P] rendered insensitive to alkaline phosphatase are plotted as a function of reaction time. A zero time background of 0.007 pmoles was subtracted from each value. The specific activity of the [5'-32~]poly(1) used was 2.95 x 104 cpmfpmole and 0.85 pmoles were added to each reaction.

1101

c

0’ N

5'-NMP

2'(3')-NMP

Alkaline

I.

1.61

1.39

3

0.06

0.21

CO.01

co.01

0.1%

[5'-32P]poly(c) and 16.67 pmoles

[5'-32P]poly(I)*poly(C) et al. (5). was used as substrate and 6.7% pmoles

JLS-V9 cell extracts were assayed according to Cranston et al. (7). with a specific activity of 1.5 x 103 cpm/pmole was used as substrate were added to each 100 ~1 reaction.

to Linne cpmlpmole

L cell extracts and T4 RNA ligase were assayed according to Walker et al. (3). [5'-32~]poly(1) with a specific activity of 1.7 x 104 cpm/pmole was used as substrate and 3.52 pmoles were added to each 100 ~1 reaction.

co.01

0.41

0.04

co.01

-T4l

+

were

Assays

+

Ligase

reaction

JLS-"93

Following

formed in the RNA ligase and methods.

0.06

co.01

f

Monophosphates L cell2

co.01

2L cell extracts were assayed according with a specific activity of 5.9 x lo3 were added to each 100 pl reaction.

1

monophosphate in materials

0.07

+

of Nucleoside

0.13

L cell1

Distribution

Isomers of nucleoside separated as described

(pmoles)

(pmoles)

hydrolysis

Enzyme source

Table

CI s 0

5'-IMP

2'(3')-IMP

Alkaline

II.

-59

c.01 .85