Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing

Abstract

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3' exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3' pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5' phosphate of the 3' fragment, which is not required for U removal. The ability of the 3' U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3' exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (Rp)alpha-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.

FIG. 1.

Accurate deletion editing of the precleaved substrate. (A) Diagram of the precleaved deletion substrate annealed to gRNA. The four overhanging U's to be removed extend above the RNA-RNA duplex. The 3" fragment contains 5" and 3" monophosphates, as shown. An asterisk indicates the [³²P]phosphate radiolabel. Hyphens have been added in gA6[14]PC-del at the ES (ES1) for clarity; the RNA strand is continuous across the ES. (B) Autoradiogram of RNA products of the precleaved deletion reaction. Reaction mixtures lacking one or more of the RNAs in panel A are labeled as follows: 5" Only, U5-5"CL only; No gRNA, U5-5"CL and 3"CL in a 1:20 molar ratio; and No 3", U5-5"CL and gA6[14]PC-del in a 1:2 molar ratio. The +ATP lane contained 300 μM ATP. All reaction mixtures except those lacking mt extract (No Ext) contained U5-5"CL, 3"CLpp, and gA6[14]PC-del in a 1:20:10 molar ratio and 3 μl of T. brucei mt extract. For clarity, the presence (+) or absence (−) of gA6[14]PC-del (gRNA) and U5-3"CLpp (3" Fragment) in each lane is indicated. The input U5-5"CL RNA is indicated with an arrow. 5" fragments with one, two, three, or four 3"-terminal U's removed are labeled −1 U through −4 U, respectively; L, ligation product of U5-5"CL with no missing U's and with U5-3"CL; E −4, edited RNA with four U's deleted. Diagrams of the editing products are also provided; the 5" and 3" fragments are represented by open and shaded rectangles, respectively, and the U's remaining at the 3" terminus of U5-5"CL are shown. An alkaline hydrolysis ladder (OH⁻) is included as a size standard.

FIG. 2.

U specificity of T. brucei mt exonuclease. The 3" end sequence of the 5" fragment included in each reaction is shown. U's exposed at the 3" terminus are underlined. Wild-type U5-5"CL was used in reaction mixtures without mt extract (No Ext) and without gRNA. Products marked −1 NT through −4 NT have 1 through 4 nucleotides removed from the 3" end, respectively; 5" fragments that have no (L) or 2 (E −2) nucleotides removed and that are ligated to 3" fragments are indicated.

FIG. 3.

Time course of U removal and precleaved deletion editing. (A) Time course of precleaved deletion editing in the presence of ATP. Reaction mixtures were incubated at 28°C for periods of time between 0 and 8 h. RNA species are designated as described for Fig. 1B. (B) Accumulation of U-removal products. The −4 U nonligated product and the −4 edited product in panel A were quantified as percentages of the total input RNA. (C) U removal without ATP. Reaction mixtures included a 3" fragment with a 5" phosphate or without a 5" phosphate (5" OH). Reaction mixtures were incubated for lengths of time between 5 min and 8 h, and the abundance of the −4 U-removal product was measured relative to the level of total input RNA. The same data are shown in the gel insets, with the input RNA (arrow) and −4 removal product indicated. Ligated products, which always totaled less than 1% of input, are not shown in the gel insets.

FIG. 4.

Protection of 3"-terminal U's from removal. Control lanes and RNA products are labeled as described for Fig. 1. (A) Products from precleaved deletion reaction mixtures containing gRNA into which zero to four guiding A's (gAs; numbers of which are indicated by the numbers above the gel) were inserted at ES1. Reactions were performed in the presence (+ATP) or absence (−ATP) of 0.3 mM ATP. (B) Protection by various guiding nucleotides. The top panel shows the locations of guiding nucleotides (lowercase letters). gA6[14]PC-del used in these reactions contained two guiding nucleotides, as indicated above each lane, reading 3" to 5" with respect to gRNA (AA in no-extract lane).

FIG. 5.

Stereospecificity of mt exoUase. Conditions, U-removal products, and ligation products are labeled as described for Fig. 1B. Reactions were carried out in the presence (+ATP) or absence (−ATP) of 300 μM ATP. U5-5"CL containing standard U residues (U) and (R_P)α-thio-U's incorporated by T7 polymerase from (S_P)α-thio-UTP [(R_P)U] was subjected to the precleaved-RNA deletion assay with U5-3"CL and gA6[14]PC-del.