Cell, Vol. 64, 667-669,

February

22, 1991, Copyright

RNA Editing: World’s Smallest

0 1991 by Cell Press

Minireview Introns?

Thomas R. Cech Department of Chemistry and Biochemistry Howard Hughes Medical Institute University of Colorado Boulder, Colorado 80309-0215

It once seemed that the nucleotide sequence of every mRNA would be a simple copy of the sequence of its DNA template. This concept suffered its first jolt in 1977 with the discovery that intervening sequences or introns interrupted many eukaryotic genes. It is now well documented that these sequences are removed at the RNA level by cleavage-ligation reactions known as RNA splicing. A second major jolt occurred with the recent finding that individual nucleotides within some mRNAs are posttranscriptionally inserted, deleted, or altered in sequenceprocesses termed RNA editing. Certain mitochondrial pre-mRNAs in the parasitic protozoa Trypanosoma, Leishmania, and Crithidia are extensively edited to give the mature, functional mRNA; U residues are added and deleted at multiple sites along the molecule (Benne et al., 1986; Feagin et al., 1987; Simpson, 1990). Guide RNAs, short RNAs that are complementary (if one allows UG wobble pairs) to edited portions of the mature mRNA, have been discovered and are thought to provide the information for this editing process (Blum et al., 1990). Sequence analysis of partially edited RNA molecules that are thought to represent intermediate steps in the process has indicated that editing involves successive cycles of U addition or deletion. It has been proposed that cycles proceed in an orderly 3’ to 5’ direction (Feagin et al., 1988; Sturm and Simpson, 1990; Van der Spek et al., 1990). Alternatively, cycles might be unordered within precisely defined domains of editing (Decker and Sollner-Webb, 1990). This minireview does not address the progression of cycles of editing along the mRNA but rather the chemical steps within a single cycle of editing. Models proposed for these events invoke a number of enzymatic activities, including an endonuclease, a terminal uridylyl transferase (TUTase), an RNA ligase, and, for the U-deletion reaction, a 3’ exonuclease (Feagin et al., 1988; Blum et al., 1990; Van der Spek et al., 1990; Simpson, 1990). The most explicit of these models is shown in Figure 1. I propose an alternative model, in which the removal of U’s is accomplished by a transesterification mechanism analogous to that of RNA splicing (Cech, 1986a), and the addition of U’s is accomplished by a reaction analogous to reverse splicing (Woodson and Cech, 1989). Thus, a single active site could perform all of RNA editing. The addition of one or more U residues to the middle of an RNA could occur as shown in Figure 2a. The 3’end of an oligo(U) sequence attacks the pm-mRNA at a position where it is mismatched with the guide sequence. In a concerted cleavage-ligation reaction, the pre-mRNA is cleaved and U’s become added to the 5’ end of a 3’-ter-

minal “half-molecule” intermediate. At this point, reversal of the reaction would regenerate the starting material. However, a new base pair can now form between the newly inserted U and the A (or G) in the guide RNA. Therefore, step 2, a pseudoreversal of the reaction with attack again occurring adjacent to the double-stranded region, leads to the addition of one U to the pre-mRNA. Such a mechanism could also allow several consecutive U’s to be added as a block. The group I and group II intron selfsplicing reactions provide precedent for ligation occurring by attack of a 3’ hydroxyl at a phosphorus atom of a standard phosphodiester linkage. It is possible that the key intermediate in this reaction scheme has already been found: Y-truncated cDNA copies of partially edited mRNA fragments often have extra Ts at their 5’ ends (Feagin et al., 1987; Abraham et al., 1988) and could correspond to the molecule labeled with the asterisk in Figure 2a. However, runs of T have also been found at the 5’ ends of cDNA copies of RNAs that are not thought to undergo editing, leading Van der Spek et al. (1990) to argue that such molecules may be artifacts of cDNA synthesis. Even if the corresponding RNA molecules exist, it would remain to be proven that they are true intermediates in editing (a shortcoming they share with all other putative editing intermediates). pre-edited

pre-mRNA

already

edited

5 cp41

I I I I I

,A” endonuclease

5’ gRNA

1

-COH

P9

I I I

I I I

,A”

TUTase

-cu

uTP PPi

PA 3J~““” ligase

,A”

ATP AMP + PPi

~

Figure 1. Model for RNA Editing by Separate Cleavage, U-Addition, and Ligation Steps, As Proposed by Slum et al. (1990) and Simpson ww Nucleotide substrate requirements expected for TUTand an RNA ligase have been added. Sold lines and letters, premRNA; lowercase u, nucleotide added by editing; gRNA, guide RNA. Interactions that hold the ?/fragment of pre-mRNA into the editing complex in the intermediate stage have been proposed but are not shown here.

Cell 668

(a) U-Addition

(b) U-Deletion

by “Splicing”

5’ --uuuoH

5’ -uulJuoH 5 Cpin pre-mRNA : /,I ’ ’ ’ ’ ’ 5 /iA gRNA

5

d:pY

;UA

I I I I I I -5

Figure 2. Model for RNA Editing terification

by Transes-

Italic U’s, oligo(U); wavy line, additional uridines or other RNA sequences (see text); three dots, possible additional base pairing prior to attack of oligo(U); l , intermediate with extra U’s attached to mRNA sequences.

--BOB * A

:Pu \OH I I I I I I I

,UA--

;ip%

%I

I I I I I

,GAUp

I I I I I

,UA---

What would be the source of the oligo(U) in such a mechanism? One possible source is the nonencoded 3’4erminal oligo(U) tail on the guide RNA (Blum and Simp son, 1998). Another might be the oligo(U) tail added to the pre-mRNA prior to polyadenylation; however, many partially edited molecules appear already to have a poly(A) tract added to their stretches of oligo(U), which would prevent the oligo(U) from serving as a U-donor (Decker and SollnerWebb, 1998). UTP could in principle be the Udonor and would provide more thermodynamic “push” for the subsequent joining reaction than would a phosphodiester bond of oligo(U). However, if the RNA molecules with more U’s at their 5’ ends than required for editing are true intermediates, then transesterification by a block of U’s rather than by UTP seems likely. The deletion of U’s from internal positions could be accomplished by the transesterification reaction shown in Figure 2b. In contrast to the U-addition reaction, where oligo(U) donates a U to an internal position in the premRNA, the oligo(U) now becomes elongated by one residue by removing it from the premFiNA. This reaction is almost but not quite the reverse of that shown in Figure 2a; in a true reverse reaction, the oligo(U) would be attacking to the 5’side of a U that was base paired, whereas in Figure 2b it is attacking 5’ to a U that is not base paired. The reactions are similar enough that they might be catalyzed by the same active site, if that active site had sufficient plasticity to recognize more than one type of pre-mRNA-guide RNA mismatch. For example, the recognition element for U-addition could be an unpaired or bulged purine on the guide RNA strand, which would cause the attack of oligo(U) to be directed opposite the unpaired purine (Figure 2a). The recognition element for U-deletion could be an unpaired or bulged U on the premRNA strand, which would cause the attack of oligo(U) to

be directed to the phosphate on the 5’side of the unpaired U (Figure 2b). This could explain the cases of 5’~CU3’sequences in pm-mRNA where the U is removed (Stuart, 1991): the G in the guide RNA might pair more stably with the C than with the U, thereby leaving the U unpaired and creating the U-deletion signal. The energy driving these reversible reactions in the directions shown in Figure 2 comes from formation of base pairs, as described previously in a model for the RNA-catalyzed replication of RNA (Cech, 1988b). Were it not for base pairing, the same U residue would undergo a nonproductive cycle of being continuously added and subtracted from the mRNA. Independent of the applicability of this particular model, there is fundamental similarity between RNA editing and RNA splicing. When a U is deleted by editing, the ‘intron” being excised is but a single nucleotide. U addition by editing is then a reverse-splicing reaction, just as group I and group II introns have been shown to undergo reverse splicing in vitro (Woodson and Cech, 1989). Sites of canonical RNA splicing reactions are often specified by guide sequences. In group,1 RNA splicing, the intron carries an internal guide sequence (Davies et al., 1982; Been and Cech, 1988); when removed from the intron, this sequence still functions as an external guide RNA (Doudna and Szostak, 1989). Group II intmns also have internal guide sequences (Jacquier and Michel, 1987) while nuclear mRNA splicing uses Ul snRNA as an external guide RNA (Mount et al., 1983). The way in which guide sequences help direct RNA splicing bears a striking resemblance to the proposed role of guide RNA in RNA editing. Thus, it seems reasonable that there could be,additional mechanistic similarities. What sort of enzymatic machinery might catalyze RNA editing? If editing does occur by a splicing mechanism, it

Minireview 669

would nevertheless be expected to use different catalytic machinery: the nucleotides that need to be recognized are clearly distinct in these two processes. The group I intron self-splicing analogy might lead one to predict editing to be RNA catalyzed. However, protein enzymes also catalyze transesterification with nucleotide and nucleic acid substrates (e.g., CID replicase, RNA ligase, polynucleotide phosphorylase, and RNA polymerase). Therefore, the mechanistic considerations do not point decisively toward catalysis by RNA as opposed to protein. In summary, the transesterification model makes strong, testable predictions about the editing reaction, as follows: l

l

l

The intermediate in each editing step would consist of a 5’ mRNA fragment terminating with a 3’ hydroxyl group, and a 3’ mRNA fragment with oligo(U) joined to its Vend by a normal 5’-+ 3’phosphodiester bond. This location of oligo(U) is opposite to that predicted by the model of Blum et al. (1990). No mRNA cleavage relevant to the processing reaction would occur without U-addition; thus, no hydrolytic enzymes (endo- or exonucleases) would be involved in editing. TUTase would not be responsible for adding U’s to sites of editing; rather it would synthesize the oligo(U) that became the source of the added Us.

Thus, if this model is incorrect, it should be disproven as authentic editing intermediates and catalytic activities are identified and characterized. For the moment it is an intriguing possibility that the actual chemistry of editing might take place in a single catalytic center that performs both U addition and U deletion. References Abraham, Been,

J. M., Feagin,

J. E., and Stuart,

M. D., and Cech,

K. (1968). Cell 55, 267-272.

T. R. (1986). Cell 47, 207-216.

Benne, R., Van Den Burg, J., Brakenhoff, J. P. J., Sloof, P., Van Boom, J. H., and Tromp, M. C. (1986). Cell 46, 819-826. Blum,

B., and Simpson,

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Davies, R. W., Waring, chio, C. (1982). Nature Decker,

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K. (1988). Cell 53, 413-422.

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339, 519-522.

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B. (1990). Cell 67, 1001-1011. W. (1989).

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RNA editing: world's smallest introns?

Cell, Vol. 64, 667-669, February 22, 1991, Copyright RNA Editing: World’s Smallest 0 1991 by Cell Press Minireview Introns? Thomas R. Cech Depar...
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