Transcripts synthesized by RNA polymerase III can be polyadenylated in an AAUAAA-dependent manner

Abstract

It is well known that nearly all eukaryotic mRNAs contain a 3' poly(A) tail. A polyadenylation signal (AAUAAA) nearby the 3' end of pre-mRNA is required for poly(A) synthesis. The protein complex involved in the pre-mRNA polyadenylation is coupled with RNA polymerase II during the transcription of a gene. According to the commonly accepted view, only RNAs synthesized by RNA polymerase II can be polyadenylated in an AAUAAA-dependent manner. Here we report the polyadenylation of short interspersed elements (SINEs) B2 and VES transcripts generated by RNA polymerase III. HeLa cells were transfected with SINE constructs with or without polyadenylation signals. The analyses of the SINE transcripts showed that only the RNAs with the AAUAAA-signal contained poly(A) tails. Polyadenylated B2 RNA was found to be much more stable in cells than B2 RNA without a poly(A) tail.

FIGURE 1.

A probable way of generation of poly(A)-containing B2 RNA, a pol III transcript of B2 SINE. (i) SINE is depicted as a rectangle; the following functional elements are shown: boxes A and B of a pol III promoter, potential polyadenylation signals (pA₁, pA₂, and pA₃), a pol III transcriptional terminator (filled box), and an oligo(A) tail (open box). Thin lines indicate DNA sequences flanking SINE including TSD (target site duplication) or short direct repeats (horizontal arrows). (ii) A 180-nt B2 RNA, a product of pol III transcription of B2 SINE. (iii) Heterogeneous in length B2 RNA, a product of polyadenylation of the 180-nt transcript. A poly(A) tail is shown as an open rectangle.

FIGURE 2.

(A) Nucleotide sequence of the mouse B2 SINE copy used for preparation of constructs. The SINE and its flanking sequences are shown in upper and lower cases, respectively. TSD flanking SINE is underlined. A pol III promoter (box A and box B), potential polyadenylation signals pA1 and pA2 (underlined), and a pol III terminator (underlined with dotted line) are indicated in the B2 sequence. (B) The structure of six constructs used in the study of the polyadenylation capability of B2 SINE pol III transcripts. The first 150 bp of B2 are depicted as a rectangle, whereas a terminal region of the B2 constructs is represented as a nucleotide sequence. Potential polyadenylation signals are underlined; a terminator is underlined with dotted line. Note that an additional T residue was introduced in the terminator, whereas an oligo(A) tail was removed from all the constructs.

FIGURE 3.

Northern blot analysis of B2 SINE transcripts isolated from HeLa cells that were transfected with B2-containing constructs with or without polyadenylation signals (see Fig 2B) as well as the construct with mutant pol III promoter (B2-mtP-pA₁pA₂). The blot analysis was performed by separating total cellular RNA by electrophoresis in an agarose (A) or polyacrylamide (B, C) gel. A180-nt B2 RNA is indicated by an arrow or brace. Longer forms of B2 RNA are marked with square brackets.

FIGURE 4.

(A) Blot analysis of RNA fractionated on oligo(dT) cellulose from HeLa cells transfected with B2 SINE constructs with (B2-pA₁pA₂) or without (B2-pA₀) polyadenylation signals. By means of chromatography on oligo(dT)-cellulose, total cellular RNA preparations were separated into poly(A)⁺ and poly(A)^– RNA fractions (A+ and A–, respectively). These RNA fractions and the total cellular RNA (Tot) including that from nontransfected cells (HeLa) were separated by electophoresis in an agarose gel followed by blot hybridization with B2 probe. (B) Removal of poly(A) tracts from B2 RNA. RNA from cells transfected with the B2-pA₁pA₂ construct (lane 1) was digested with RNAse H following incubation with (lane 3) or without (lane 2) oligo(dT)_12–18. A B2 RNA was detected by Northern hybridization following PAGE. A 180-nt and long B2 RNA are indicated by brace and square brackets, respectively.

FIGURE 5.

(A) Consensus of nucleotide sequences of 20 cloned cDNAs corresponding to the 3′ end of the B2 RNA (a rectangle depicts the rest of cDNA sequence). RNA was isolated from HeLa cells transfected with a B2-pA₁pA₂ construct, and B2 RNA-derived cDNAs were synthesized, cloned, and sequenced. Noteworthy are terminator sequence shortening (TCTTT) and the emergence of poly(A). (B) Diagram showed distribution of the numbers of B2 cDNA clones analyzed against length of poly(A) segments in them. The number of residues in the poly(A) segments from each clone was counted and numbers of the clones with poly(A) segment length fallen into each size range were summarized.

FIGURE 6.

(A) Blot analysis of the RNA from HeLa cells transfected with construct B2-pA₁pA₂ and treated with actimomycin D, cordycepin, α-amanitin, or neither of the inhibitors (control). An RNA from untransfected cells (HeLa) or cells transfected with construct B2-pA₀ was also analyzed for comparison. Hybridization with the 5S rRNA probe was used as a sample-loading control. The short-lived histone H1F1 mRNA was detected by hybridization in order to verify the inhibition of pol II by α-amanitin. Normalized data on H1F1 mRNA hybridization indicate an 80% inhibition of pol II. (B) Study of B2 RNA stability in HeLa cells transfected with construct B2-pA₁pA₂ or B2-pA₀. The RNA was isolated in indicated time periods following addition of actinomycin D to transfected cells.

FIGURE 7.

(A) A nucleotide sequence of the bat VES SINE copy used for preparation of constructs. The SINE and its flanking sequences are shown in upper and lower cases, respectively. TSD flanking SINE is underlined. In the VES sequence, a pol III promoter (box A and box B), a potential polyadenylation signal pA (underlined), and a pol III terminator (underlined with dotted line) are indicated. (B) The structure of two constructs used to study the polyadenylation ability of VES SINE pol III transcripts. The first 200 bp of VES are depicted as a rectangle, whereas a terminal region of the SINE constructs is represented as a nucleotide sequence. A potential polyadenylation signal is underlined; a terminator is underlined with a dotted line. Note that two additional T residues were introduced in the terminator of the both constructs. (C) Blot analysis of VES SINE transcripts isolated from HeLa cells transfected with VES-containing constructs with (VES-pA₁) or without (VES-pA₀) a polyadenylation signal. Long forms of VES RNA were indicated with a square bracket.