Gösta Winberg

Full-length RNA-seq from single cells using Smart-seq2

Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1–3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA−) RNA.

Smart-seq2 for sensitive full-length transcriptome profiling in single cells

Single-cell gene expression analyses hold promise for characterizing cellular heterogeneity, but current methods compromise on either the coverage, the sensitivity or the throughput. Here, we introduce Smart-seq2 with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells. Smart-seq2 transcriptome libraries have improved detection, coverage, bias and accuracy compared to Smart-seq libraries and are generated with off-the-shelf reagents at lower cost.

Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma

The BamHI fragment containing the Epstein-Barr virus (EBV) LMP1 gene was cloned from a genomic library of the nude mouse-propagated Chinese nasopharyngeal carcinoma CAO. The sequence of the LMP1 gene and its promoter and enhancer was determined. The nucleotide sequence of the CAO isolate differed from those of the B95-8 and Raji isolates in the promoter/enhancer region; the amino acid sequence of the protein also differed. Structural differences in the protein were located mainly in the 20 N-terminal residues and the array of repeated amino acids in the C-terminal part of the protein, in which the CAO isolate displays a cluster of seven perfect repeats of 11 amino acids (aa). Three of these repeats have no counterpart in the other virus strains. This, together with two deletions of five and 10 aa in the C-terminal part, yields a protein of 404 aa, compared to 386 aa for B95-8 and Raji. The larger LMP1 protein was detected on immunoblots of tissue samples from the CAO nude mouse tumour, and was also present in EBV-negative B cell lines and immortalized keratinocytes transfected with the cloned gene. A XhoI restriction site in exon 1 of the B95-8 BNLF-1 gene was absent from the CAO EBV isolate, as well as from 36 of 37 Chinese NPC biopsies tested. In contrast, 17 of 19 NPC biopsies of African origin retained this XhoI site.

WW Domains Provide a Platform for the Assembly of Multiprotein Networks

ABSTRACT WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs and phosphorylated serine/threonine-proline sites. To pursue the functional properties of WW domains, we employed mass spectrometry to identify 148 proteins that associate with 10 human WW domains. Many of these proteins represent novel WW domain-binding partners and are components of multiprotein complexes involved in molecular processes, such as transcription, RNA processing, and cytoskeletal regulation. We validated one complex in detail, showing that WW domains of the AIP4 E3 protein-ubiquitin ligase bind directly to a PPXY motif in the p68 subunit of pre-mRNA cleavage and polyadenylation factor Im in a manner that promotes p68 ubiquitylation. The tested WW domains fall into three broad groups on the basis of hierarchical clustering with respect to their associated proteins; each such cluster of bound proteins displayed a distinct set of WW domain-binding motifs. We also found that separate WW domains from the same protein or closely related proteins can have different specificities for protein ligands and also demonstrated that a single polypeptide can bind multiple classes of WW domains through separate proline-rich motifs. These data suggest that WW domains provide a versatile platform to link individual proteins into physiologically important networks.

Latent Membrane Protein 2A of Epstein-Barr Virus Binds WW Domain E3 Protein-Ubiquitin Ligases That Ubiquitinate B-Cell Tyrosine Kinases

ABSTRACT The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling. The N-terminal cytoplasmic region of LMP2A has multiple tyrosine residues that upon phosphorylation bind the SH2 domains of the Syk tyrosine kinase and the Src family kinase Lyn. The LMP2A N-terminal region also has two conserved PPPPY motifs. Here we show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including AIP4 and KIAA0439, and demonstrate that AIP4 and KIAA0439 form physiological complexes with LMP2A in EBV-positive B cells. In addition to a C2 domain and four WW domains, these proteins have a C-terminal Hect catalytic domain implicated in the ubiquitination of target proteins. LMP2A enhances Lyn and Syk ubiquitination in vivo in a fashion that depends on the activity of Nedd4 family members and correlates with destabilization of the Lyn tyrosine kinase. These results suggest that LMP2A serves as a molecular scaffold to recruit both B-cell tyrosine kinases and C2/WW/Hect domain E3 protein-ubiquitin ligases. This may promote Lyn and Syk ubiquitination in a fashion that contributes to a block in B-cell signaling. LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signaling.

Tn5 transposase and tagmentation procedures for massively scaled sequencing projects

Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large-scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from subpicogram amounts of cDNA. The comparison of single-cell RNA-sequencing libraries generated using produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, because naked Tn5 can be annealed to any oligonucleotide of choice, for example, molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enable innovation in sequencing-based applications.

Epstein-Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue

Epstein-Barr virus (EBV)-encoded LMP1 gene derived from a nude mouse passaged nasopharyngeal carcinoma (NPC) of Chinese origin (C-LMP1) and its B cell (B95-8 prototype)-derived counterpart (B-LMP1) were compared for their ability to induce tumour rejection in a mouse mammary adenocarcinoma system. Each of the two LMP1 genes was introduced individually by retroviral vectors into a non-immunogenic mammary carcinoma line, S6C, that originated in an ACA (H-2f) mouse. Syngeneic ACA mice were immunised for 3 consecutive weeks with irradiated B- or C-LMP1 expressors or control cells. The immunised and control mice were then challenged with graded numbers of viable cells from the corresponding cell line. Only the B-LMP1 expressing cells were highly immunogenic. Up to 10(5) cells were rejected in pre-immunised mice, whereas at least 10(2) cells grew in non-immunised controls. No rejection response was detected against the C-LMP1 expressing cells which grew equally well in control and immunised mice, with a minimum inoculum of 10(2) cells in the majority of the clones. In a previous study, we found numerous sequence differences between B- and C-LMP1. The question of whether any of these differences is related to the non-immunogenicity of C-LMP1 needs further investigation. Meanwhile, our findings raise the possibility that the NPC cells may escape host rejection by the development of a non-immunogenic LMP1 variant under the impact of immunoselection.

Encapsidation of adenovirus 16 DNA is directed by a small DNA sequence at the left end of the genome

Abstract We have identified a DNA sequence in adenovirus type 16 which contains recognition signals for encapsidation of the viral DNA. The sequence acts in cis to direct the encapsidation of DNA from the end of the viral genome where it is located. The sequence is normally contained in the first 390–400 bp of the left end of the genome. The location was determined by analyzing a series of spontaneous mutants of Ad16 which carried reduplications of 200 to >500 bp of left end sequences at the right end of the genome, thus giving rise to enlarged inverted terminal repetitions (ITR). In plaque-purified (PP) Ad16 prototype virus the subgenomic DNA found in incomplete virus particles exclusively represents left end sequences. When the reduplication mutants were analyzed, we found that a reduplication of about 390 bp enabled subgenomic DNA molecules containing the right end to be encapsidated into incomplete particles as well. A reduplication of about 290 bp, however, did not allow subgenomic DNA containing the right end to be encapsidated. The difference in encapsidation described could not be attributed to an asymetric DNA replication in the mutants, since subgenomic DNA originating from both ends of the genome was produced in equal amounts in the infected cells. We conclude that an essential part of the encapsidation sequence must be located between 290 and 390 bp from the left end of the Ad16 genome.

Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines

Ubiquitin specific proteases (USPs) regulate the production and recycling of ubiquitin and are thereby critically involved in the control of cell growth, differentiation, and apoptosis. Increasing evidence implicates deregulation of USPs in malignant transformation but there is very little information on the overall and specific activity of USPs in normal and tumor tissues. We have used a chemistry‐based functional proteomics approach to profile the activities of individual USPs in biopsies of human papillomavirus (HPV) carrying cervical carcinoma and adjacent normal tissue. To assess the contribution of HPV proteins, USP activity was also compared in HPV positive and negative cervical carcinoma cell lines and HPV E6/E7 immortalized human keratinocytes. The activity of the C‐terminal hydrolases UCH‐L3 and UCH37 was upregulated in the majority of tumor tissues compared to the adjacent normal tissues. UCH‐L1 activity was lower in a significant proportion of the tumors but to a less extent in advanced tumors. In accordance with the relatively low UCH‐L1 activity in tumor biopsies, UCH‐L1 was detected only in one out of eight cervical carcinoma lines. UCH‐L1, UCH‐L3, USP7, and USP9X activity was upregulated following HPV E6/E7 immortalization of keratinocytes, suggesting a role of these enzymes in growth transformation.

Differences in the growth pattern and clinical course of EBV-LMP1 expressing and non-expressing nasopharyngeal carcinomas

All low differentiated or anaplastic forms of nasopharyngeal carcinoma (NPC) carry multiple copies of EBV-DNA and express EBNA1. The major membrane protein, LMP1, is only expressed in 65% of the tumours. The physiological function of LMP1 in the viral life cycle is unknown, but it has been shown to transform established rodent fibroblasts and immortalised human keratinocytes in vitro, and to increase the likelihood of a malignant transformation. We studied 74 cases collected from the Shanghai and Guanzhou areas in China. LMP1 expression was assessed in tumour biopsies by immunoblotting. Clinical and follow-up data were evaluated according to the classification of WHO. The laboratory and the clinical data were assembled in a mutually independent double blind fashion. Our findings indicate that the LMP1-positive tumours grew faster and more expansively than LMP1-negative tumours, but nevertheless had a better prognosis. LMP1-negative tumours recurred at a higher frequency, and showed an increased tendency to metastasise.