The F2 USE stimulates mRNA expression and 3′ end formation in a position- and sequence-dependent manner. (A) Schematic representation of the HBB (human β-globin)—F2 hybrid gene construct with a tandem array of two F2 3′ end formation signals used in transient transfection experiments (Ter: stop codon). The F2 USE was either completely or partially replaced by an unrelated nucleotide sequence, or displaced downstream and upstream of its original position as depicted. (B) In vivo assay carried out by transient transfection of a HBB-F2 hybrid gene construct with modifications of the USE as depicted in (A). The bar diagram shows the fold difference of the mRNA ratio processed at the 5′ or the 3′ site (5′/3′) relative to the F2 WT construct (highlighted)±s.e. (at least four independent experiments). (C) In vivo assay carried out by transient transfection of a HBB-F2 hybrid gene construct with modifications of the USE as depicted. The bar diagram shows the fold difference of the mRNA ratio processed at the 5′ or the 3′ site (5′/3′) relative to the F2 WT construct (highlighted)±s.e. (at least four independent experiments).

The F2 USE specifically interacts with nuclear proteins. (A) EMSA carried out with a F2 USE containing 21-mer RNA oligonucleotide (lane 1, free probe) after incubation in HeLa nuclear extract (NE, lanes 2–11) or cytoplasmatic extract (S100, lanes 12 and 13), respectively. Specificity of the RNA–protein interaction is shown by coincubation of an unlabeled F2 USE-containing 21-mer RNA oligonucleotide as cold competitor (lanes 6–9), of an unrelated competitor (lane 10) and a competitor, including the hFip1 binding site of the L3 RNA (lane 11). (B) UV crosslinking study carried out with the same USE-containing or competitor RNA oligonucleotides (lane 0, free probe) after incubation in HeLa nuclear extract (NE, lanes 1–11 (lane 1 without UV light exposure)) or cytoplasmatic extract (S100, lanes 12 and 13), respectively.

USE-binding splicing factors specifically promote the expression of USE-containing mRNAs. (A) Representative immunoblots of protein lysates obtained from cells treated with siRNAs directed against USE-binding proteins (lane 1, each panel). Lanes 2–5 in each panel show a serial reduction of the load of protein lysates obtained from cells treated with a luciferase siRNAs as control for nonspecific RNAi effects and for quantification of the RNAi efficiency. (B) Schematic representation of the HBB-F2 hybrid gene construct with a tandem array of two 3′ end formation signals. The F2 USE of the 5′ located 3′ end processing signal was either maintained (‘5′USE' construct) or completely replaced by an unrelated nucleotide sequence (‘no USE' construct). (C) In vivo assay carried out by transient transfection of a HBB-F2 hybrid gene construct, with or without a USE (B), after depletion of USE-binding proteins (A). Each number represents the fold difference of the mRNA ratio processed at the 5′ or the 3′ site (5′/3′) relative to the respective ratio in the odd numbered lanes (representative figure of three independent experiments).

Model for USE-dependent RNA processing at (non-canonical) 3′ end formation signals. 3′ End processing of USE-containing mRNAs is proposed to depend on the formation of USE-dependent RNP complexes that participate in an extensive molecular network coordinating gene expression. USE-binding splicing factors are depicted as positive effectors (green complex), potentially involving a direct stimulation of PAP via U2AF65. The USE-binding protein complex (Table I) is indicated by gray shading. The USE-binding proteins are proposed to bridge and promote splicing and 3′ end processing. Specifically, the 65 kDa subunit of the U2AF dimer may link 3′terminal exon definition with efficient 3′ end processing. On the other hand, the USE-dependent RNP complexes establish the link to the 3′ end processing machinery by interaction with CPSF 100 and CstF 77. The polypyrimidine tract-binding protein PTB/hnRNPI and U2AF65 likely represent direct USE-interacting proteins and may thus nucleate the USE-dependent RNP complexes for efficient stimulation of 3′ end formation.

The F2 USE is highly conserved among higher eukaryotes. (A) Sequence comparison of the 3′ ends of vertebrate F2 genes (encompassing the entire 3′ UTR until the poly(A) signal). Shaded sequences denote identity. The graphical representation of the nucleic acid multiple sequence alignment (shown below) highlights the sequence conservation of the F2 USE according to the WebLogo 3 algorithm (Materials and methods), which contains a composite of two highly conserved sequence motifs (TATTTTTGT, highlighted; Xie et al, 2005). (B) Applying a sequence search algorithm that takes into consideration both the strand specificity and the typical length distribution for 3′ UTR motifs (peak >8-mers after exclusion of miRNAs target sites; Xie et al, 2005), more than 1700 human transcripts were identified to contain the nonameric USE core sequence motif. Number of hits are shown according to the localization of the sequence element within the mRNAs (bar diagram, x-axis, 5′ to 3′, left to right). Positive hits were filtered according to their relative location with respect to the poly(A) signal (AATAAA and ATTAAA, respectively). The identity of transcripts that contained the USE core sequence in close proximity to the poly(A) signal (<30 nucleotides upstream of the poly(A) signal (90 and 61 transcripts upstream of the AATAAA or ATTAAA, respectively)) is depicted in Supplementary Tables I and II.

Affinity purification followed by mass spectroscopy identifies proteins that specifically interact with the F2 USE. (A) Silver-stained SDS–PAGE polyacrylamide gel of protein samples derived from affinity purification with immobilized 3′ biotinylated 21-mer RNA oligonucleotides with the F2 USE motif (USE, lanes 1 and 5), with a mutated F2 USE motif (USEmut, lane 2) or with an unrelated sequence context (Unrel., lane 3). Lanes 1–3 show protein samples eluted with up to 2000 mM NaCl (starting concentration 150 mM NaCl). Lane 4 shows the autoradiograph of a UV crosslink that highlights the size of direct interaction partners of the F2 USE (dotted arrows indicate putative direct USE-binding proteins that could not be unequivocally assigned by the mass spectrometry data) in comparison to the band pattern of directly and indirectly interacting proteins derived from affinity purification (lane 1). Silver-stained bands in lane 1 were cut out and subjected to mass spectrometry (protein names are only indicated at the respective size where the respective peptide score was maximal; canonical 3′ end processing factors are highlighted in yellow; p54nrb peptides of unexpected small size are highlighted in light gray; for mass spectrometry data, see also Table I). The analysis also revealed the presence of ATP citrate synthase, LRP 130, HSP 90- and tubulin (asterisks), which were judged to represent likely contaminants and were not included in Table I. (B) Immunoblots of eluates from affinity purifications for proteins identified by mass spectrometry (Table I). Lanes 1–3 correspond to samples as indicated in Figure 4A. Additional information on controls for unspecific RNA–protein interaction and equal loading is available in Supplementary Figure S2.

Depletion of the USE-binding protein hnRNPI and U2AF65 specifically reduces the mRNA expression of USE-containing endogenous transcripts. (A) In vivo RNA–protein interaction assay based on mRNA analyses in RNP IPs carried out with antibodies as indicated. IPs were carried out with HUH-7 cell lysates after UV crosslinking (lanes 1–6), FA crosslinking (lanes 7–12) or without crosslinking (lanes 13–18; for further information see Materials and methods). The F2 mRNA was analyzed by RT–PCR (primer dimers are indicated by asterisks); lanes 19 and 20 represent positive and negative controls, respectively. (B) In vivo RNA–protein interaction assay based on reporter mRNA analysis in IP samples derived from FA crosslinked HeLa cells transfected with reporter constructs containing either a F2 USE (USE reporter), a mutated F2 USE (USEmut reporter) or an unrelated sequence context (Unrel. reporter, see Supplementary Figure S2). (C) Endogenous F2, BCL2L2, IVNS, ACTR and C2 mRNA, and ACTG1, HPRT, CBFB and MAP3K mRNA abundance of HUH-7 cells after RNAi directed against USE-binding proteins as indicated (x-axis). The fold change of mRNA expression upon candidate siRNA treatment (y-axis) is quantified relative to the mRNA expression of cells treated with luciferase siRNAs after normalization against endogenous ACTB mRNA. Each bar represents values of at least five independent RNAi experiments determined by quantitative RT–PCR in duplicates (±s.e.).