TbU1-70K is a U1 snRNP-specific protein and binds specifically to the 5′ loop sequence of U1 snRNA

<p><b>Copyright information:</b></p><p>Taken from "U1 small nuclear RNP from : a minimal U1 snRNA with unusual protein components"</p><p>Nucleic Acids Research 2005;33(8):2493-2503.</p><p>Published online 29 Apr 2005</p><p>PMCID:PMC1087902.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Comparison of the domain structures of (Tb08.4A8.530) and the human U1-70K (A25707) proteins. () Western blot analysis of U1 snRNP proteins. U1 snRNPs were affinity-purified from extract by a 2′--methyl RNA antisense oligonucleotide, protein was prepared and analyzed by SDS–PAGE and western blotting, using polyclonal rabbit antibodies against TbU1-70K (U1-70K) or non-immune serum (NIS). The arrow points to the immunostained TbU1-70K band of apparent molecular weight 40 kDa. Protein markers are on the right (in kDa). () U1 snRNA is specifically coprecipitated from extract by anti-Tb U1-70 antibodies. Immunoprecipitations were carried out from extract, using NIS, or with antibodies against the TbU1-70K protein (U1-70K) or against the trypanosome Sm proteins (Sm). RNA was purified from the immunoprecipitates and analyzed by 3′ end labeling with [P]pCp. The positions of the SL RNA and snRNAs are marked on the right. , P-labeled pBR322/HpaII markers. () RNA from the same immunoprecipitates was also analyzed by primer extension with a U1-specific oligonucleotide. In addition, RNA from a 10% aliquot of the input was included; the positions of the primer () and the U1-specific primer-extension product (U1) are marked on the right. , P-labeled pBR322/HpaII markers. () P-labeled U1 snRNA and mutant derivatives [as indicated above the lanes; see (F)] were transcribed and incubated with GST-TbU1-70K, followed by GST pull-down. For each reaction, 10% of the input () and the total precipitated material () were analyzed. , P-labeled pBR322/HpaII markers. () Sequences and proposed secondary structures of the U1 snRNA and its mutant derivatives. The boxed sequence in the U1 snRNA indicates the Sm site; the two arrows indicate a potential second stem–loop. Below, the sequences of the stem–loop derivatives are given; the circled nucleotides mark the two positions in the human loop that differ from the sequence, and the single-nucleotide mutation (A21) in the mutant human loop

Protein–protein interactions in the trypanosome U1 snRNP: TbU1-70K interacts with both TbU1C and TbU1-24K

<p><b>Copyright information:</b></p><p>Taken from "U1 small nuclear RNP from : a minimal U1 snRNA with unusual protein components"</p><p>Nucleic Acids Research 2005;33(8):2493-2503.</p><p>Published online 29 Apr 2005</p><p>PMCID:PMC1087902.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () GST-fusion proteins of TbU1C, TbU1-24K and TbU1-70K, as well as GST alone as a control were immobilized on glutathione-Sepharose. Corresponding aliquots of immobilized proteins were analyzed for their protein content by SDS–PAGE and Coomassie staining. The arrows point to the proteins listed above the lanes. , protein marker (in kDa). () Immobilized GST proteins (as indicated above the lanes) were incubated with S-labeled TbU1C (lanes 1–4), TbU1-24K (lanes 5–8) or TbU1-70K (lanes 9–12). After washing, bound proteins were recovered and analyzed by SDS–PAGE and fluorography. The arrows point to the respective S-labeled proteins

U1C (TbU1C): a U1 snRNP-specific component binding specifically to the 5′ terminal sequence of U1 snRNA

<p><b>Copyright information:</b></p><p>Taken from "U1 small nuclear RNP from : a minimal U1 snRNA with unusual protein components"</p><p>Nucleic Acids Research 2005;33(8):2493-2503.</p><p>Published online 29 Apr 2005</p><p>PMCID:PMC1087902.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () ClustalW alignment of the protein sequences for the newly identified U1C homologs from , and , in comparison with the human U1C sequence. The conserved CH-type Zn finger within the boxed sequence is highlighted by large-size letters; asterisks indicate absolutely conserved amino acid positions. Accession numbers (GeneDB): (Tb10.70.5640), (Tc00.1047053511367.354) and (LmjF21.0320); human U1C (P09234). () Extract was prepared from a cell line, which stably expresses TAP-tagged TbU1C protein, and used to affinity-purify TAP-tagged complexes. Purification was followed by analyzing copurifying RNAs by northern blotting, using a mixed snRNA probe (snRNA positions indicated on the right). , DIG marker V (Roche). Lane 1, 1% of input; lane 2, 10% of IgG-selected and TEV-released material. Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. () TbU1C protein binds specifically to the 5′ terminal sequence of U1 snRNA. GST TbU1C protein was incubated with P-labeled full-length U1 snRNA (lanes 1 and 2) and various U1 snRNA derivatives: U1 Δstem–loop (lanes 3 and 4), U1 Δ5′(1–14) (lanes 5, 6), U1 Δ5′(1–30) (lanes 7 and 8), U1 5′ stem–loop (lanes 9 and 10), U1 5′(1–14) (lanes 11 and 12) or a 17mer control RNA (lanes 13 and 14). In each case, 10% of the input () and the total GST pull-down material () were analyzed

The polyadenylation complex of <i>Trypanosoma brucei:</i> Characterization of the functional poly(A) polymerase

<p>The generation of mature mRNA in the protozoan parasite <i>Trypanosoma brucei</i> requires coupled polyadenylation and <i>trans</i> splicing. In contrast to other eukaryotes, we still know very little on components, mechanisms, and dynamics of the 3′ end-processing machinery in trypanosomes. To characterize the catalytic core of the polyadenylation complex in <i>T. brucei</i>, we first identified the poly(A) polymerase [Tb927.7.3780] as the major functional, nuclear-localized enzyme in trypanosomes. In contrast, another poly(A) polymerase, encoded by an intron-containing gene [Tb927.3.3160], localizes mainly in the cytoplasm and appears not to be functional in general 3′ end processing of mRNAs. Based on tandem-affinity purification with tagged CPSF160 and mass spectrometry, we identified ten associated components of the trypanosome polyadenylation complex, including homologues to all four CPSF subunits, Fip1, CstF50/64, and Symplekin, as well as two hypothetical proteins. RNAi-mediated knockdown revealed that most of these factors are essential for growth and required for both <i>in vivo</i> polyadenylation and <i>trans</i> splicing, arguing for a general coupling of these two mRNA-processing reactions.</p

Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

<p>Circular RNAs (circRNAs) were recently described as a novel class of cellular RNAs. Two circRNAs were reported to function as molecular sponges, sequestering specific microRNAs, thereby de-repressing target mRNAs. Due to their elevated stability in comparison to linear RNA, circRNAs may be an interesting tool in molecular medicine and biology. In this study, we provide a proof-of-principle that circRNAs can be engineered as microRNA sponges. As a model system, we used the Hepatitis C Virus (HCV), which requires cellular microRNA-122 for its life cycle. We produced artificial circRNA sponges <i>in vitro</i> that efficiently sequester microRNA-122, thereby inhibiting viral protein production in an HCV cell culture system. These circRNAs are more stable than their linear counterparts, and localize both to the cytoplasm and to the nucleus, opening up a wide range of potential applications.</p

Model of intra-U1 snRNP U1-70K/U1C cross-regulation.

<p>Binding of intact U1 snRNPs to three cryptic 5′ splice sites (labeled with A, B, C) within intron 7 of the U1-70K pre-mRNA (middle) activates an alternative 3′ splice site. Inclusion of the alternative exon 7a introduces a premature termination codon (stop sign) into the mature U1-70K mRNA, which is degraded by nonsense-mediated decay (NMD; following the pathway upwards). Reduced U1-70K mRNA and protein levels result in a co-depletion of U1C protein; thus, U1 snRNPs are assembled inefficiently. U1C/U1-70K-deficient U1 snRNPs are unable to activate the alternative 3′ splice site, therefore, constitutive U1-70K splicing is enhanced, and more functional U1-70K mRNA and protein are produced (following the pathway downwards). Normal U1 snRNP assembly is restored and alternative 3′ splice site activation can occur again to close the regulatory circle (for a detailed description, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#s3" target="_blank"><i>Discussion</i></a>).</p

U1C-dependent activation of an alternative 3′ splice site within U1-70K introduces a PTC: RNA-Seq analysis.

<p>(<b>A</b>) Read-density maps derived from RNA-Seq analysis of control- (blue) and U1C-siRNA-treated (ΔU1C, red) HeLa cells are shown above the exon-intron structure of the human U1-70K gene (exons 1–10 indicated as black boxes, with the narrower parts representing the untranslated regions at the 5′ and 3′ ends). Below, the conservation in vertebrates is given in green. The dashed region is shown in more detail below in panel B. (<b>B</b>) Read densities of the control- (blue) and U1C-knockdown HeLa cells (ΔU1C, red) for the U1C-dependent alternatively spliced and highly conserved exons 7–8 region. (<b>C</b>) Quantitation of the use of the alternative 3′ splice site in intron 7, as determined by specific junction-read numbers given above and below each exon-intron structure (in blue for control-, in red for U1C-knockdown). The green box indicates the potential alternative exon 7a generated by use of the alternative 3′ splice site at position +642 in intron 7, which introduces a premature termination codon (stop sign), and one of downstream cryptic 5′ splice sites (labeled A, B, C, and D).</p

U1C depletion results in specific alternative splicing alterations in HeLa cells: Specificity and validation.

<p>(<b>A</b>) U1C knockdown (kd) in HeLa cells. Whole cell lysates were analyzed by SDS-PAGE and Western blot detecting U1C and γ-tubulin. U1 snRNA steady-state levels were analyzed by Northern blotting with probes specific for U1 snRNA and, as a loading control, U3 snoRNA. HeLa cells after U1C knockdown (ΔC) and luciferase-siRNA treated control cells (ctr) were compared. (<b>B</b>) Graphical overview of U1C-dependent alternative splicing targets identified by RNA-Seq analysis. (<b>C</b>) U1 snRNA blocking in HeLa cells. The efficiency of U1 snRNA blocking was determined by RNase H protection and silver staining. The positions of the full-length U1 snRNA (U1 uncut), the RNase H-cleaved U1 snRNA (U1 cut), and the U2 snRNA (as a control) are marked on the right. (<b>D</b>) Alternative splicing patterns of selected U1C target genes (names above the lanes) were analyzed by RT-PCR, using total RNA from HeLa cells after U1C-knockdown (ctr vs. ΔC) or U1 snRNA blocking (ctr vs. U1). Target-specific primers (arrows in the schematics on the right of the panels) were designed to amplify both alternative splicing isoforms. <i>M</i>, DNA size markers (in bp). Upper panel: Top and lower bands represent exon inclusion and skipping products, respectively; an unspecific product for <i>SNHG5</i> is marked by open circles between the lanes. Lower panel: For <i>MARCH7</i> top and lower bands reflect usage of the proximal and distal 5′ splice site, respectively. For <i>UFM1</i> three alternative 5′ splice sites are activated upon U1C knockdown labeled with 1, 2, and 3 on the right.</p

Alternative 3′ splice site activation requires U1 snRNP binding to downstream cryptic 5′ splice sites.

<p>(<b>A</b>) U1-70K minigene constructs used for <i>in vivo</i> splicing analysis (see panel B), including exons 7, 7a, and 8; the arrows indicate the primers used for RT-PCR analysis. The enlargement below represents the region covered by the biotinylated transcripts used for <i>in vitro</i> binding studies (see panel C). The exact positions of the stop codon (dashed vertical line) and the three cryptic 5′ splice sites (bold vertical lines labeled with A, B, and C) are shown together with the 5′ splice site sequences, including all point mutations analyzed. The two solid lines above the enlarged exon mark the positions of the splice site blocking antisense morpholinos (see panel D); their labeling (AB and BC AMO) refers to the 5′ splice site they block. (<b>B</b>) Mutational analysis of U1-70K alternative splicing. Splicing patterns of U1-70K minigenes (as indicated) in control- (ctr) and U1C-knockdown (ΔC) HeLa cells were analyzed by RT-PCR, detecting alternative 3′ splice site activation (primers 7-7a; top panel), exon 7a inclusion and skipping (primers 7–8; middle panel), and as a loading control, exon 7 alone (bottom panel); splicing products are depicted on the right. Percentages of exon 7a inclusion are given below with standard deviations calculated from three individual experiments [n = 3]; the labels within the second panel indicate, which cryptic 5′ splice sites were used in each case for 7a inclusion, with letters in parentheses marking the less frequently used splice sites (see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen.1003856.s003" target="_blank">Figure S3B</a></b>). (<b>C</b>) U1 snRNP binds to cryptic 5′ splice sites of U1-70K exon 7a. 3′-biotinylated RNAs spanning “exon 7a” including flanking intronic sequences (as shown enlarged in the middle of the schematic in panel A) were incubated with HeLa nuclear extracts (2% input). Bound proteins (2/3 of selected material) were analyzed by Western blotting, using antibodies against U1-70K, U1A, and U1C; bound U1 snRNA was detected by Northern blot hybridization. (<b>D</b>) Antisense morpholino (AMO) transfection in HeLa cells. In two separate assays HeLa cells were transfected with AMOs either against the cryptic 5′ splice sites of U1-70K exon 7a (AB, BC; left panel) or the 5′ end of U1 snRNA (U1; right panel), or with an unspecific control morpholino (ctr). Cells were either left untreated (−CHX) or were treated with cycloheximide (+CHX) before RT-PCR analysis. The label within the middle panel indicates which cryptic 5′ splice sites are used for exon7a inclusion, with letters in parantheses marking the less frequently used splice sites. M, DNA size markers (in bp).</p

U1-70K mRNA and protein levels are upregulated in human and zebrafish upon loss of U1C.

<p>(<b>A</b>) Exon-intron structure of U1-70K exons 7–8. The predominant splicing patterns in control and in U1C-knockdown HeLa cells are depicted by dashed lines above and below the schematic, respectively. The grey shading downstream of “exon 7a” indicates that the 7a-8 junction is not detectable in control cells without cycloheximide treatment. Arrows represent the RT-PCR primers used in (B) and (D) to detect alternative 3′ splice site activation (primers 7-7a), exon 7a inclusion, and exons 7–8 splicing. (<b>B</b>) U1-70K alternative splicing after U1C knockdown. HeLa cells were treated with two different siRNAs against U1C (ΔC or ΔC*, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#s4" target="_blank">Materials and Methods</a>), or a control siRNA (ctr), comparing untreated cells (−CHX) or cells after treatment with cycloheximide (+CHX). Alternative splicing patterns were analyzed by RT-PCR. The label within the middle panel indicates which cryptic 5′ splice sites are used for exon7a inclusion, with letters in parentheses marking the less frequently used splice sites (see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen.1003856.s003" target="_blank">Figure S3A</a></b>). β-actin serves as an internal loading control. (<b>C</b>) U1-70K protein levels after U1C knockdown in HeLa cells. Whole cell lysates or nuclear extracts were analyzed by SDS-PAGE and Western blot, detecting U1-70K, γ-tubulin (as a loading control), and U1C. The fold change of U1-70K protein expression after U1C knockdown is given below (ΔU1C/ctr). (<b>D</b>) Add-back of U1C restores normal U1-70K splicing in HeLa and zebrafish (<i>D. rerio</i>). Left panel: Control- (ctr) and U1C-knockdown (ΔC) HeLa cells are compared with U1C-knockdown cells expressing Flag/HA-tagged U1C. Knockdown and over-expression were verified by Western blot analysis. Right panel: A wildtype zebrafish embryo (wt) is compared with two U1C-knockout mutants, without (mut) and with ZfU1C-cRNA injection (rsc = rescue). In both assays, alternative splicing patterns were analyzed by RT-PCR, and β-actin serves as an internal loading control. The rescue effect was confirmed by Western blot analysis of single-embryo lysates. M, DNA size markers (in bp).</p