Exonic sequences in the 5' untranslated region of alpha-tubulin mRNA modulate trans splicing in Trypanosoma brucei

Abstract

Previous studies have identified a conserved AG dinucleotide at the 3' splice site (3'SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei alpha-tubulin 3'SS region is required to specify accurate 3'-end formation of the upstream beta-tubulin gene and trans splicing of the downstream alpha-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3'SS identification. Our results indicate that a minimal alpha-tubulin 3'SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by the trans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the alpha-tubulin 3'SS is dependent upon the presence of exon sequences. Furthermore, beta-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace alpha-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the alpha-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar to cis-splicing enhancers described in other systems.

FIG. 1

Schematic representation of a portion of the β-tubulin–α-tubulin gene cluster of T. brucei. Open boxes indicate the β- and α-tubulin-coding regions, which are not drawn to scale. The thin line indicates the 145-nt-long intergenic region between the β-tubulin poly(A) sites or A1 sites and the AG dinucleotide at the α-tubulin 3′SS. The positions of the long and short pPy tracts are indicated, as is the position of the putative branch sites (BS). SL and A flags mark the positions of SL and poly(A)-site additions, respectively.

FIG. 2

cis competition between duplicated α-tubulin 3′SSs. (A) Schematic representation of plasmid constructs used for transfection. WTS⁺ contains the β- and α-tubulin sequences from the nucleotide after the β-tubulin termination codon to the nucleotide preceding the α-tubulin ATG. These sequences are sandwiched between the GFP- and CAT-coding regions. The ribosomal promoter, indicated by a flag upstream, directs synthesis of the pre-mRNA. An SmaI site (S) was introduced by site-directed mutagenesis at position 474 of the β-tubulin–α-tubulin intergenic region (14) of the parent plasmid to generate WTS⁺. In AS1 the minimal 3′SS of α-tubulin mRNA, from 4 nt upstream from the branch sites (BS) to and including the AG dinucleotide, was duplicated at the SmaI site of WTS⁺. In AS2 the duplicated region included the 5′UTR of α-tubulin mRNA to the nucleotide preceding the ATG translation initiation codon. SM8 is a mutant derivative of AS2 in which the 5′ half of the long pPy tract of the duplicated 3′SS was mutagenized as described in Materials and Methods. wt3′SS and d3′SS represent the wt and duplicated 3′SSs, respectively. Filled squares and circles indicate the usage of the 3′SS and poly(A) sites, respectively, whereas open squares and circles indicate that the 3′SS and poly(A) sites, respectively, are not used. The identities of the other symbols are as described in the legend to Fig. 1. Arrows below the GFP- and CAT-coding regions indicate the approximate positions of gene-specific oligonucleotides which were used for 5′- and 3′-end RACE analyses. Results of 5′-end RACE (B) and 3′-end RACE (C) analyses of transcripts produced by transient expression of the constructs diagrammed in panel A are shown. Arrows indicate the positions of the amplified DNA fragments diagnostic of usage of the duplicated or wt 3′SS and of the A1 to 3 polyadenylation sites. Lane M, MspI digest of pBR322 DNA as a marker. Representative molecular sizes (in base pairs) are shown. Lane −, RACE products obtained with RNA from mock-transfected cells.

FIG. 3

Analysis of 3′SS use by primer extension analysis. RNAs from cells transfected with the construct indicated above each lane were reverse transcribed by using as a primer a ³²P-labeled oligonucleotide complementary to nt 26 to 39 of the CAT-coding region. The positions of the primer extension products diagnostic of use of the duplicated 3′SS (d3′SS) or wt 3′SS are indicated. Open circles indicate primer extension products whose origins are uncertain (see the text for details). Sizes are indicated in nucleotides.

FIG. 4

Effects of 5′UTR truncations on the relative use of duplicated and wt3′SSs. (A) Schematic representations of the AS2 parent construct and of the 5′UTR deletion derivatives containing either 21 (UTR21), 42 (UTR42), 63 (UTR63), or 84 (UTR84) nt of the 106-nt-long α-tubulin 5′UTR. Results of 5′-end RACE (B) and 3′-end RACE (C) analyses of RNAs from transfected cells are shown. The sizes of the RACE products diagnostic of duplicated 3′SS use varied because of the various sizes of the 5′UTR portion included in each construct. Lane M, MspI digest of pBR322 DNA as a marker. Representative molecular sizes (in base pairs) are shown. Lane −, RACE products obtained with RNA from mock-transfected cells. d3′SS, duplicated 3′SS.

FIG. 5

Northern analysis of CAT and GFP transcripts in cells transfected with the constructs shown in Fig. 4A. The asterisks indicate the positions of putative dicistronic transcripts.

FIG. 6

Effects of block substitutions on the relative use of the duplicated and wt α-tubulin 3′SSs. (A) Schematic representations of the parent AS2 construct and of the mutagenized SM derivatives. Substituted nucleotides are indicated by filled boxes. Results of 5′-end RACE (B) and 3′-end RACE (C) analyses of RNA from transfected cells are shown. The identity of each construct is indicated above each lane. Lane M, MspI digest of pBR322 DNA as a marker. Representative molecular sizes (in base pairs) are shown. Lane −, RACE products obtained with RNA from mock-transfected cells. (D) Sequences of the α-tubulin 3′SS acceptor and 5′UTR, which was duplicated in the AS2 mutant. The nucleotide changes in mutants SM1 to -8 are indicated in lowercase letters. Dots represent unchanged nucleotides. Filled circles above the sequence indicate the putative branch site adenosines. d3′SS, duplicated 3′SS.

FIG. 7

Effects of replacement of the α-tubulin 5′UTR with the actin or β-tubulin 5′UTR or unrelated sequences. (A and B) Schematic representations of the various constructs used for transfection. ACTsub contains the 55-nt-long actin 5′UTR in place of the α-tubulin 5′UTR; βTUBsub contains the 59-nt-long β-tubulin 5′UTR. In RADsub the α-tubulin 5′UTR was replaced with a 100-nt-long fragment derived from the plasmid vector pCR^TMII. In ACTsubD and βTUBsubD the actin and β-tubulin 5′UTR substitutions of ACTsub and βTUBsub replaced the corresponding regions of the duplicated 3′SS of plasmid AS2. The positions of the SL addition and poly(A) sites for the RADsub constructs are only indicative and were not precisely determined. Symbols are as defined in the legend of Fig. 2A. ACT, actin; αTUB, α-tubulin; βTUB, β-tubulin; d3′SS, duplicated 3′SS. Results of 5′-end RACE (C) and 3′-end RACE (D) analyses of RNA from transfected cells are shown. Lane M, MspI digest of pBR322 DNA as a marker. Representative molecular sizes (in base pairs) are shown. Lane −, RACE products obtained with RNA from mock-transfected cells. (E and F) Northern blot analysis of CAT and GFP transcripts in cells transfected with the constructs shown in Fig. 7A and B. Asterisks indicate the positions of putative dicistronic transcripts.