The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism.

IntroductionEfficient delivery of genes into primary human B lymphocytes would allow the investigation of gene functions in these cells for the purposes of research and the development of gene therapies. One could then test in mature B cells the promoters/genes potentially suitable for stem cell-based therapies for immunodeficiencies. 1 Vectors achieving the efficient transfection of primary B cells would most likely also be suitable for the delivery of genes into freshly collected B tumor cells-for example, for the development of immune-based anti-B-tumor therapies. 2 Various viral vectors are currently being studied for their ability to transduce hematopoietic cells. [3][4][5] Retroviral vectors derived from murine leukemia virus (MLV) 6,7 can transfer genes into immortal human B-cell lines, such as lymphoblastoid cells, 8 and primary B precursors, 9 but they are inefficient for mature human B cells. 10,11 These simple retroviruses can transduce genes only into actively dividing cells, 12 but a potent T-independent mitogen for human B cells in vitro, such as lipopolysaccharide (LPS) for murine B cells, has not been found. 13 In addition, MLV vectors might not be well adapted for human B cells because of the host species difference. HIV-1 and HIV-derived pseudotyped lentiviral vectors efficiently integrate into human cells, irrespective of cell division. 14-22 High transgene expression from such vectors in human T cells or total lymphocytes has been reported. [23][24][25] Generally, productive HIV infection or lentivectormediated transduction of truly quiescent lymphocytes has not been observed; activation, at least from G 0 to G 1 , seems to be required. 23,[25][26][27][28] Efficient transduction of primary acute lymphoblastic leukemia cells with a bicistronic HIV vector, leading to the expression of a cytokine (granulocyte macrophage-colonystimulating factor [GM-CSF]) and an immunostimulatory molecule (CD80), has also been achieved, 29 indicating a potential use of such vectors in novel anti-B-tumor therapies.In this study we investigated the transduction of peripheral blood B cells with multiply attenuated HIV vectors pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein. 21 Efficient transduction of such B cells occurred after their activation in a culture system using murine EL-4 B5 thymoma cells as helper T cells in conjunction with human cytokines. [30][31][32][33] This system leads to proliferation and subsequent plasmocytic differentiation of all naive and memory human B subsets. 33 Nondividing, freshly isolated multiple myeloma cells were also efficiently transduced by HIV vector. By contrast, an MLV vector pseudotyped with VSV G protein was inefficient even in dividing B cells. F.B. and P.S. contributed equally to this work. D.T. has declared a financial interest as consultant to Cell Genesis, a company whose potential product is related to the HIV-1 vectors used in this study.Reprints: R. Zubler, Division of Hematology, University Hospital, 1211 Geneva-14, Switzerland; e-mail: rudolf.zubl...

The heterodimeric subunit, SRP9/14, of the signal recognition particle (SRP) has previously been found to bind to scAlu and scB1 RNAs in vitro and to exist in large excess over SRP in anthropoid cells. Here we show that human and mouse SRP9/14 bind with high affinities to other Alu-like RNAs of different evolutionary ages including the neuron-specific BC200 RNA. The relative dissociation constants of the different RNA-protein complexes are inversely proportional to the evolutionary distance between the Alu RNA species and 7SL RNA. In addition, the human SRP9/14 binds with higher affinity than mouse SRP9/14 to all RNAs analyzed and this difference is not explained by the additional C-terminal domain present in the anthropoid SRP14. The conservation of high affinity interactions between SRP9/14 and Alu-like RNAs strongly indicates that these Alu-like RNPs exist in vivo and that they have cellular functions. The observation that human SRP9/14 binds better than its mouse counterpart to distantly related Alu RNAs, such as recently transposed elements, suggests that the anthropoid-specific excess of SRP9/14 may have a role in controlling Alu amplification rather than in compensating a defect in SRP assembly and functions.

The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP). The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle. SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA. We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide. We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure. Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio. In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay. Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities. The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro.

+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel crossreactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.

No abstract

The anti-apoptotic proteins bcl-2 and bcl-x L seem to exhibit strictly opposite expression patterns in normal lymphoid cell differentiation stages, with bcl-2 low and bxl-x L high in immature and mature proliferating cells, the reverse being the case in recirculating quiescent cells. However, it is in fact not known whether recirculating memory cells are bcl-x L low or high. We analyzed memory (immunoglobulin isotype-switched) B cells in human peripheral blood, which were small lymphocytes in the G0 phase of the cell cycle, but proliferated better than naive B cells in response to Staphylococcus aureus Cowan I. Ex vivo these cells co-expressed bcl-2 together with bcl-x L mRNA and protein at high levels. The mcl-1 mRNA level was low. The bcl-x L mRNA level decreased during culture in medium containing fetal calf serum, which implies that it is maintained in vivo by continuous or frequent, non-mitogenic signal(s). The high bcl-x L expression of memory B cells may be relevant with regard to their longevity and/or their capacity to undergo an accelerated secondary type immune response.

The anti-apoptotic proteins bcl-2 and bcl-xL seem to exhibit strictly opposite expression patterns in normal lymphoid cell differentiation stages, with bcl-2 low and bxl-xL high in immature and mature proliferating cells, the reverse being the case in recirculating quiescent cells. However, it is in fact not known whether recirculating memory cells are bcl-xL low or high. We analyzed memory (immunoglobulin isotype-switched) B cells in human peripheral blood, which were small lymphocytes in the G0 phase of the cell cycle, but proliferated better than naive B cells in response to Staphylococcus aureus Cowan I. Ex vivo these cells co-expressed bcl-2 together with bcl-xL mRNA and protein at high levels. The mcl-1 mRNA level was low. The bcl-xL mRNA level decreased during culture in medium containing fetal calf serum, which implies that it is maintained in vivo by continuous or frequent, non-mitogenic signal(s). The high bcl-xL expression of memory B cells may be relevant with regard to their longevity and/or their capacity to undergo an accelerated secondary type immune response.