Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor

Abstract

The mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.

Figure 1.

Engineering a U1 snRNA dependent expression system using the HIV unspliced env mRNA. (A) Diagram of the pHIV-Env model construct. An HIV U3 region promoter (grey box) drives transcription through R and U5 regions, all three of which make up the long terminal repeat (LTR). The cDNA has the SD1/SA4b intron removed and contains two exons, shown in black rectangles and one intron bounded by SD4 and SA7. The Env (gp160) open reading frame (ORF) expressed from the unspliced mRNA is shown, the Rev ORF expressed from spliced mRNA is also shown, Vpu and Nef ORFs are present but do not express significant protein in this spliced isoform. (B) Mutations made to SD4 shown in red, a second cryptic SD SD4x overlaps the minimal upstream ORF (uORF) and Vpu start. (C) Use of SD4x was prevented by mutations shown in red at the 3′ splice site, SA7. The branch point (BP), polypyrimidine tract (PPT) and cryptic acceptors were mutated in addition to SA7 itself. All mutations introduced were silent with respect to the Env-coding sequence. (D) Splice site mutations ablated splicing. RT–PCR using Odp.2 and Odp.40 was performed on RNA extracted from cells transfected with the indicated mutant. PCR products were extracted from the gel, cloned and sequenced. Sequence of SA7x is shown to the right. (E) The spliced product of the pHIV-Env transcript makes the Rev protein which controls the export of unspliced 4-kb mRNA and hence Env protein expression. The amino acid sequence is shown at the SD4/SA7 exon/exon junction. The Rev ORF was inactivated using a stop codon at Rev amino acid 38. This mutant was assessed by northern blotting of 4-kb (unspliced) and 2-kb (spliced) mRNA present in nuclear (N), nuclear-associated rough endoplasmic reticulum (R) and cytoplasmic (C) fractions. The 4- and 2-kb probe was made using Odp.1409 and Odp.1410. Probes were designed to bind to gapdh (Acc: NM_002046) for loading, U6 snRNA (Acc: X07425), 7SL (Acc:NR_002715) and mitochondrial tRNA^lys (Acc:X93334, tRNA 14) to control for the efficiency of N, R and C fractions respectively.

Figure 2.

U1 snRNA enhances transcription independent of splicing (A) Hydrogen bonding (H-bonding) of U1 snRNA to SD4. The 5′arm of U1 snRNA contacts SD4 through 16 hydrogen-bonds (H-bonds), while mutSD4 makes only nine H-bonds to U1 snRNA. H-bonding was recovered by co-transfection of a modified U1 snRNA mutated (shown in large bold font) to make 17 H-bond contacts with mutSD4. (B) U1mut5′arm rescues nuclear accumulation of mutSD4SA7 sufficient to rescue protein expression in the absence of splicing. Cells were transfected with 2 µg of pHIV-Env with the indicated mutations, 20 ng of pCMV-Tat, 100 ng of pCMV-Rev and 1.5 µg of pUCB-U1 with the indicated modification to the 5′arm. These cells were either fractionated and RNA analysed by northern blotting for 4- and 2-kb mRNA and RT–PCR for 2-kb cDNA or they were lysed and protein analysed by western blotting for Env (gp160). U1mut5′arm was also detected in the various RNA fractions using RT–qPCR with primers specific to the mutated 5′ arm and normalisation to gapdh mRNA as described in the methods. Lanes are as follows, Lane 1—Mock, Lane 2—WT.R38 + WT 5′ arm, Lane 3—WT.R38 + mut 5′ arm. Lane 4—mutSD4SA7 + WT 5′ arm, Lane 5—mutSD4SA7 + WT 5′ arm. (C) U1 snRNA does not enhance mRNA stability. Transcription was inactivated with 5 µg/ml of Actinomycin D and 4-kb mRNA levels from mutSD4SA7 in the presence of U1 snRNA WT 5′arm (−U1) and U1 snRNA mut5′arm (+U1) assessed by RT–QPCR. 4-kb mRNA levels were normalized to the geometric mean of three reference genes as described in the methods. Error bars represent standard deviation of the mean from three transfections performed on different days.

Figure 3.

U1 snRNA enhancement of transcription requires the Sm domain and stem loop II (A) U1 snRNA secondary structure and location of U170k, U1A, U1C and Sm proteins in the small ribonucleoprotein (snRNP). Mutations to disrupt binding of U170k, U1A, Sm are shown in red as well as the deletion made to stem loop II in blue. (B) Sm domain and stem loop II are important for U1 snRNA rescue. Wild-type U1 snRNA and the four U1 snRNA body (nt 12–164) mutants with either a wild-type or 5′arm (nt 1–11) mutant were co-transfected with a gp140uncGFP reporter (shown) containing the SD4SA7 mutation in addition to 20 ng of pCMV-Tat and 100 ng of pCMV-Rev. Values represent the mean of the fold difference between the wild-type and mut5′arm from four transfections performed on different days. Error bars represent the standard deviation of the mean.

Figure 4.

The requirement for U1 snRNA binding is independent of promoter and an upstream artificial intron. (A) The promoter does not alter the requirement for U1 snRNA recruitment. pHIV-Env and pCMV-Env are shown with their promoter, 5′ exon and intron sequence drawn to scale. Cells were transfected with these constructs containing either the WT.R38 or mutSD4SA7 mutations, 20 ng of pCMV-Tat for the pHIV construct and 100 ng of pCMV-Rev, harvested for protein and Western blotted for Env (gp160) and β-actin. (B) An upstream artificial intron cannot rescue protein or mRNA expression from mutSD4SA7. pCMV-Intron-Env is shown drawn to scale relative to pHIV-Env and pCMV-Env. Cells were transfected with either WT.R38 or mutSD4SA7 along with 100 ng of pCMV-Rev. The northern probe was made using Odp.1126 and Odp.92 (C) U1 snRNA rescue of mutSD4SA7 is independent of the promoter and an upstream intron. The GFP reporter from Figure 3B was expressed from pCMV-Intron as shown. Values represent the GFP fluorescence from the reporter shown above, normalized to WT.R38 plus U1 5′arm WT. The Sm U1 snRNA mutant was used as a negative control. Error bars represent the standard deviation of the mean for three transfections performed on different days.

Figure 5.

U1 snRNA transcriptional enhancement depends on in the intron sequence following the U1-bound SD. (A) Plasmid map of the GFP and GFP(SD4SA7) constructs. (B) The SD4-SA7 intron is efficiently and accurately spliced from GFP(SD4SA7) to produce GFP protein. Fluorescence from GFP, GFP(SD4SA7) and GFP(mutSD4SA7) constructs was measured using FACS. GFP(mutSD4SA7) contains the mutations outlined in Figure 1B and C and these transfections included 100 ng of pCMV-Rev. (C) U1 snRNA increases the nuclear accumulation of unspliced mRNA in the absence of HIV exonic sequence. Cells were transfected with the indicated constructs, including 100 ng of pCMV-Rev, and fractionated into nuclear (N), nuclear-associated rough ER (R) and cytoplasmic (C) fractions. RNA was northern blotted using a probe to GFP made from Odp. 445 and Odp.1250. The purity of fractions was assessed by probing for U6 snRNA. (D) Nuclear unspliced mRNA band intensities were quantified from the blot shown above and normalized to the band intensities of gapdh. Values represent the fold difference in mRNA levels expressed compared to GFPi plus U1WT5′arm.

Figure 6.

Tat acetylation is important for transcription in the absence of U1 snRNA recruitment. (A) Tat and not Rev partially replaced the need to bind U1. gp140uncGFP with indicated mutations reported a dose-response from pCMV-Tat and pCMV-Rev. Values represent the percentage expression level of mutSD4SA7 relative to WT.R38. (B) Post-translational modifications of Tat and locations of acetylation sites. Lysine 51 (K51) in the RNA-binding domain is acetylated by p300. (C) Lysine 51 is important only for expression in the absence of U1 snRNA recruitment by splicing signals. Tat mutant K51A was tested for trans-activation activity by co-transfection with WT.R38 or mutSD4SA7. Protein expression was assessed by FACS analysis of gp140uncGFP. Error bars represent the standard deviation of the mean of five independent transfections.

Figure 7.

Proposed model for U1 snRNA enhancement of transcription. (A) Transcription of pre-mRNA containing the SD4-SA7 intron is inhibited in the absence of U1 snRNA recruitment by SD4. (B) When U1 snRNA binds at the SD, transcription through the SD4-SA7 intron is efficient, possibly due to an increase in the processivity of elongation via P-TEFb phosphorylation of the Pol II CTD at Serine 2.