Binding of hnRNP LL is more sensitive than hnRNP L to spacing of CA dinucleotides.

<p>(A) Complete sequence used for binding in panel B and as backbone for experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080701#pone-0080701-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080701#pone-0080701-g005" target="_blank">5</a>. (B-F) Representative native gel analysis (EMSA) of purified, recombinant GST-tagged hnRNP L or Flag-tagged hnRNP LL incubated with radiolabeled T7-transcribed RNAs corresponding to a variant of the sequence in panel A. For each panel the sequence shown was substituted at the capitalized positions in panel A. CA dinucleotides are underlined, for panels C-F spacers between CAs are in red (N=spacer #). Affinities (Concentration of 50% binding = BC₅₀) from at least three independent replicates of the EMSAs are shown below gels. Bound and free probe is indicated to the right of each gel. Asterisks indicate BC₅₀s which are significantly different from CACACA (*p<0.05, **p<0.01), as calculated by a unpaired t-test between binding values determined from a minimum of three independent protein titrations.</p

Complete RNAcompete data shows a hnRNP LL-specific preference for a two nucleotide spacer between CA dinucleotides.

<p>A bargraph for hnRNP L (A) and hnRNP LL (B) showing the mean Z-score for each possible spacing of 0 to 4 between two CA dinucleotides. Means were calculated for all 8-mers within the RNAcompete dataset that contain the sequence CA[N_0-4]CA. Error bars show the standard error.</p

NS1-BP interacts with hnRNPs that directly bind M1 mRNA.

<p>(A) The entire M1 RNA was radiolabeled uniformly at either C (left) or U (right) residues, incubated with nuclear extract under splicing conditions, crosslinked with 254 nm light and digested with RNase. The reaction was then either resolved by SDS-PAGE (10% Total) or incubated in separate reactions with the antibodies indicated (IP: anti-). Immunoprecipitated proteins were resolved by SDS-PAGE. Position of molecular weight markers are also shown. Vertical dotted line shows that the samples were loaded in the same gel but not next to each other. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003460#ppat.1003460.s004" target="_blank">Figure S4</a> shows immunoblot analysis of nuclear extract with antibodies against NS1-BP and hnRNP U, which did not directly bind M1 mRNA but are present in the extract. (B) A549 cells were infected with A/WSN/33 at MOI 5 for 5 h and were then cross-linked with 0.3% formaldehyde. Cells were lysed with RIPA buffer, and the lysates were immunoprecipitated with control IgG or NS1-BP antibodies. After a series of washes, the cross-link was reversed, and associated RNAs were isolated. Real-time quantitative RT-PCR was used to measure M1 mRNA. Housekeeping genes (GAPDH, β-actin, and α-tubulin) were included to test the specificity of the assay. Data was normalized to the viral M segment. The graph displays the mean ± SEM (n = 3). Comparison between cross-linked samples shows significant (p<0.05) differences between association of NS1-BP with M1 mRNA and the lack of association of NS1-BP with the 3 host mRNAs tested.</p

Domain architecture and conservation of hnRNP L and hnRNP LL.

<p>Domain structure of hnRNP L (top) or hnRNP LL (bottom) showing total protein size and relative location and size of RNA-recognition motifs (RRMs) and linker regions (grey) including the glycine-rich (G) and proline-rich (P) segments of hnRNP L. Percent amino acid identity for each domain of hnRNP L compared to the respective domain of hnRNP LL is given between protein schematics.</p

Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

<div><p>Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA₃, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.</p></div

HnRNP L and hnRNP LL have different sensitivity to mutation of individual CA dinucleotides within the CD45 exon 4 ESS1 element.

<p>(A) Sequence of the ESS1 element from CD45 exon 4 with ARS element underlined, CA dinucleotides in bold and numbered consecutively, and segments that correspond to the above-described binding consensus for hnRNP L (L) or hnRNP LL (LL) are indicated in blue and grey respectively. (B) Binding curve for hnRNP L, as calculated from a minimum of three EMSAs, for the wildtype ESS1 or variants that are mutated at individual CAs (see panel D for legend). (C) Binding curve for hnRNP LL as described in panel B. (D) Legend for binding curves in panels B and C along with the apparent affinities for each RNA-protein combination as derived from the binding curves. Asterisks indicate affinities (concentration of 50% binding = BC₅₀) which are significantly different from ESS (* p<0.05, ** p<0.01), as calculated by a unpaired t-test.</p

NS1-BP interacts with RNA-binding proteins, RNA polymerase II, and influenza A virus polymerase.

<p>(A) Top panel, HeLa cell lysates were immunoprecipitated with control IgG or NS1-BP antibodies. Interacting proteins were resolved by SDS-PAGE and identified by mass spectrometry. Bottom panel, A549 cells were treated with non-targeting or NS1-BP siRNAs (1, 2, and 3) for 48 h. Cell extracts were subjected to immunoblot analysis, which shows NS1-BP knockdown. β-actin served as loading control. (B) Immunoprecipitation was performed with control IgG or anti-NS1-BP antibodies, in the absence or presence of RNase A. Western blots were then performed with the depicted antibodies, selected based on the proteins identified in A. (C) A549 cells were mock-infected or infected with influenza virus at MOI 5 for 5 h. Cells were lysed and subjected to size exclusion chromatography. The fractions were concentrated by TCA precipitation and analyzed by western blot with the indicated antibodies. (D) A549 cells were mock-infected or infected with influenza virus (A/WSN/33) at MOI 2 for 16 h. Cell lysates were immunoprecipitated with control IgG or anti-NS1-BP antibodies. Western blots were performed with the depicted antibodies.</p

hnRNP K is required for optimal M2 protein production and influenza A virus replication.

<p>A549 cells were transfected with non-targeting or hnRNP K siRNAs for 48 h prior to infection. siRNA transfected cells were infected with A/WSN/33 at (A) MOI 2 or (B) MOI 0.001. Cells were harvested at the indicated hours post-infection, and viral protein accumulation was assessed by immunoblot analysis. Each protein band in (A) and (B) was quantified by ImageJ and normalized to α-tubulin levels. (C) Control or hnRNP K siRNA transfected cells were infected with A/WSN/33 at MOI 0.001. At 36 hours post-infection, cell supernatants were collected and subjected to viral titer analysis (n = 3) or (D) ATP level analysis to determine cell viability (n = 3, representative experiment). Error bars denote mean + SEM. *p<0.05.</p

Position-dependent activity of CELF2 in the regulation of splicing and implications for signal-responsive regulation in T cells

CELF2 is an RNA binding protein that has been implicated in developmental and signal-dependent splicing in the heart, brain and T cells. In the heart, CELF2 expression decreases during development, while in T cells CELF2 expression increases both during development and in response to antigen-induced signaling events. Although hundreds of CELF2-responsive splicing events have been identified in both heart and T cells, the way in which CELF2 functions has not been broadly investigated. Here we use CLIP-Seq to identified physical targets of CELF2 in a cultured human T cell line. By comparing the results with known functional targets of CELF2 splicing regulation from the same cell line we demonstrate a generalizable position-dependence of CELF2 activity that is consistent with previous mechanistic studies of individual CELF2 target genes in heart and brain. Strikingly, this general position-dependence is sufficient to explain the bi-directional activity of CELF2 on 2 T cell targets recently reported. Therefore, we propose that the location of CELF2 binding around an exon is a primary predictor of CELF2 function in a broad range of cellular contexts.</p

hnRNP K regulates M1 mRNA splicing.

<p>(A) Cell extracts from A549 cells transfected with non-targeting siRNAs or siRNAs that target various indicated hnRNPs for 48 h were subjected to immunoblot analysis with antibodies specific to each hnRNP. (B and C) A549 cells were transfected as in B and then infected with A/WSN/33 at MOI 2 for the indicated time points. In C, NS1-BP and hnRNP K were simultaneously knocked down. Total RNA was isolated at each depicted time point and analyzed by real-time RT-PCR with primers specific to M1 and M2 mRNAs. M2/M1 mRNA ratios are shown. Error bars represent mean ± SD (n = 3). *p<0.05, **p<0.01, ***p<0.001.</p