Journal: Nature Communications

Article Title: An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal

doi: 10.1038/ncomms10950

Figure Lengend Snippet: ( a ) Domain organization of full-length (fl) U2AF 65 and constructs used for RNA binding and structural experiments. The N- and C-terminal residue numbers are indicated. An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF 65 1,2 and dU2AF 65 1,2L constructs. ( b ) Comparison of the apparent equilibrium affinities of various U2AF 65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). The flU2AF 65 protein includes a heterodimerization domain of the U2AF 35 subunit to promote solubility and folding. The apparent equilibrium dissociation constants ( K D ) for binding the AdML 13mer are as follows: flU2AF 65 , 30±3 nM; U2AF 65 1,2L, 35±6 nM; U2AF 65 1,2, 3,600±300 nM. ( c ) Comparison of the RNA sequence specificities of flU2AF 65 and U2AF 65 1,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. The K D 's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF 65 , 41±2 nM; U2AF 65 1,2L, 31±3 nM. The K D 's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF 65 , 414±12 nM; U2AF 65 1,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a . The average apparent equilibrium affinity ( K A ) and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t -tests with Welch's correction are indicated as follows: ** P <0.01; NS, not significant, P >0.05. The purified protein and average fitted fluorescence anisotropy RNA-binding curves are shown in . RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif.

Article Snippet: The U2AF 65 1,2L FRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF 65 residues 113–343.

Techniques: Construct, RNA Binding Assay, Binding Assay, Solubility, Sequencing, Two Tailed Test, Purification, Fluorescence

Journal: Nature Communications

Article Title: An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal

doi: 10.1038/ncomms10950

Figure Lengend Snippet: ( a ) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF 65 1,2L structures. The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus. For clarity, we consistently number the U2AF 65 1,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. The prior dU2AF 65 1,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF 65 RRM1 and RRM2 by crystallographic symmetry). Italics, disordered in the structure. ( b ) Stereo views of a ‘kicked' 2| F o |−| F c | electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF 65 1,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). ( c ) Cartoon diagram of this structure. Crystallographic statistics are given in and the overall conformations of U2AF 65 1,2L and prior dU2AF 65 1,2/U2AF 65 1,2 structures are compared in . BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose.

Article Snippet: The U2AF 65 1,2L FRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF 65 residues 113–343.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal

doi: 10.1038/ncomms10950

Figure Lengend Snippet: New residues of the U2AF 65 1,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow. The nucleotide-binding sites of the U2AF 65 1,2L and prior dU2AF 65 1,2 structure are compared in . The first and seventh U2AF 65 1,2L-binding sites are unchanged from the prior dU2AF 65 1,2–RNA structure and are portrayed in . The four U2AF 65 1,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in . The representative U2AF 65 1,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: ( a ) rU2 of structure iv; ( b ) rU3 of structure iii; ( c ) rU4 of structure i; ( d ) rU5 of structure iii; ( e ) rU6 of structure ii; ( f ) dU8 of structure iii; ( g ) dU9 of structure iii; ( h ) rC9 of structure iv. ( i ) Bar graph of apparent equilibrium affinities ( K A ) of the wild type (blue) and the indicated mutant (yellow) U2AF 65 1,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). The apparent equilibrium dissociation constants ( K D ) of the U2AF 65 1,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average K A and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t -tests with Welch's correction are indicated as follows: ** P <0.01; * P <0.05; NS, not significant, P >0.05. The average fitted fluorescence anisotropy RNA-binding curves are shown in .

Article Snippet: The U2AF 65 1,2L FRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF 65 residues 113–343.

Techniques: Mutagenesis, Binding Assay, Two Tailed Test, Fluorescence, RNA Binding Assay

Journal: Nature Communications

Article Title: An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal

doi: 10.1038/ncomms10950

Figure Lengend Snippet: ( a ) Contacts of the U2AF 65 inter-RRM linker with the RRMs. A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue). Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. Other linker residues are coloured either dark blue for new residues in the U2AF 65 1,2L structure or light blue for the remaining inter-RRM residues. The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right). ( b ) Bar graph of apparent equilibrium affinities ( K A ) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF 65 1,2L protein compared with mutations of the residues shown in a : 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF 65 1,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF 65 1,2L (residues 141–237, 258–342). The apparent equilibrium dissociation constants ( K D ) of the U2AF 65 1,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF 65 1,2L, 123±5 nM; dU2AF 65 1,2, 5000±100 nM; 3Mut, 5630±70 nM. The average K A and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t -tests with Welch's correction are indicated as follows: ** P <0.01; * P <0.05; NS, not significant, P >0.05. The fitted fluorescence anisotropy RNA-binding curves are shown in . ( c ) Close view of the U2AF 65 RRM1/RRM2 interface following a two-fold rotation about the x -axis relative to a .

Article Snippet: The U2AF 65 1,2L FRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF 65 residues 113–343.

Techniques: Mutagenesis, Two Tailed Test, Fluorescence, RNA Binding Assay

Journal: Nature Communications

Article Title: An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal

doi: 10.1038/ncomms10950

Figure Lengend Snippet: ( a ) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract ( py ) or the strong AdML Py tract ( PY ) (sequences inset). ( b ) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF 65 or a triple U2AF 65 mutant (3Mut) of Q147A, R227A and V254P residues. ( c ) A bar graph of the average percentage of the py -spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF 65 added; white, WT U2AF 65 ; grey, 3Mut U2AF 65 ). The average percentages and s.d.'s are given among four independent biological replicates. **** P< 0.0001 for two-tailed unpaired t -test with Welch's correction. Protein overexpression and qRT-PCR results are shown in .

Article Snippet: The U2AF 65 1,2L FRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF 65 residues 113–343.

Techniques: Construct, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Two Tailed Test, Over Expression, Quantitative RT-PCR

Journal: Nature Communications

Article Title: An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal

doi: 10.1038/ncomms10950

Figure Lengend Snippet: ( a , b ) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF 65 1,2L RRMs bound to a Py-tract oligonucleotide ( a , representative structure iv ) or ‘closed' NMR/PRE-based model of U2AF 65 1,2 ( b , PDB ID 2YH0) in identical orientations of RRM2. The U2AF 65 1,2L FRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. ( c – f , i , j ) The U2AF 65 1,2L FRET (Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni +2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands ( c , d ), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) ( e , f ), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) ( i , j ). In g and h , the immobilization protocol was reversed. The untethered U2AF 65 1,2L FRET (Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). Typical single-molecule FRET traces ( c , e , g , i ) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). Additional traces for untethered, RNA-bound U2AF 65 1,2L FRET (Cy3/Cy5) are shown in . Histograms ( d , f , h , j ) show the distribution of FRET values in RNA-free, slide-tethered U2AF 65 1,2L FRET (Cy3/Cy5) ( d ); AdML RNA-bound, slide-tethered U2AF 65 1,2L FRET (Cy3/Cy5) ( f ); AdML RNA-bound, untethered U2AF 65 1,2L FRET (Cy3/Cy5) ( h ) and adenosine-interrupted RNA-bound, slide-tethered U2AF 65 1,2L FRET (Cy3/Cy5) ( j ). N is the number of single-molecule traces compiled for each histogram.

Article Snippet: The U2AF 65 1,2L FRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF 65 residues 113–343.

Techniques: Microscopy, Variant Assay, Fluorescence

Journal: Nature Communications

Article Title: An extended U2AF 65 –RNA-binding domain recognizes the 3′ splice site signal

doi: 10.1038/ncomms10950

Figure Lengend Snippet: ( a ) Diagram of the U2AF 65 , SF1 and U2AF 35 splicing factors bound to the consensus elements of the 3′ splice site. A surface representation of U2AF 65 1,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. MDS-relevant mutated residues of U2AF 65 are shown as yellow spheres (L187 and M144). ( b ) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF 65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF 65 1,2L crystal structures. Alternatively, a conformation of U2AF 65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF 65 population towards the ∼0.45 FRET value. RRM1, green; RRM2, pale blue; RRM extensions/linker, blue.

Article Snippet: The U2AF 65 1,2L FRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF 65 residues 113–343.

Techniques: Sequencing, Binding Assay, RNA Binding Assay