The capacity of highly parallel sequencing technologies to detect small RNAs at unprecedented depth suggests their value in systematically identifying microRNAs (miRNAs). However, the identification of miRNAs from the large pool of sequenced transcripts from a single deep sequencing run remains a major challenge. Here, we present an algorithm, miRDeep, which uses a probabilistic model of miRNA biogenesis to score compatibility of the position and frequency of sequenced RNA with the secondary structure of the miRNA precursor. We demonstrate its accuracy and robustness using published Caenorhabditis elegans data and data we generated by deep sequencing human and dog RNAs. miRDeep reports altogether approximately 230 previously unannotated miRNAs, of which four novel C. elegans miRNAs are validated by northern blot analysis.

The translocation site (translocon), at which nascent polypeptides pass through the endoplasmic reticulum membrane, contains a component previously called 'signal sequence receptor' that is now renamed as 'translocon-associated protein' (TRAP). Tbo glycosylated subunits of the TRAP complex have been identified before (a and P subunits). We now show that the TRAP complex is actually comprised of four membrane proteins (a, P, y, 4, present in a stoichiomemc relation, which are genuine neighbours in intact microsomes. The amino acid sequences of the additional, nonglycosylated subunits were deduced from cloning of the corresponding cDNAs. The 6 subunit spans the membrane only once and has its major portion, containing a disulfide bridge, at the lumenal side. The y subunit is predicted to span the membrane four times.Proteins are translocated across the endoplasmic reticulum (ER) membrane at specific sites (translocons [l]), probably through protein-conducting channels (for review see 121). To identify membrane proteins located in the vicinity of translocating polypeptides, crosslinking methods have been applied [3 -91. Short translocating polypeptide chains, representing an early phase of the translocation process, can be crosslinked through their signal sequence to an integral, glycosylated membrane protein that is about 35 kDa and has a cytoplasmic tail of about 5 kDa [3]. Based on these properties, the 'signal sequence receptor' (a subunit) was purified [lo] and received its name on the assumption that it was identical with the major crosslinking partner of short nascent chains. However, it is now clear that both this protein and the recently discovered 'translocating-chain-associating membrane' (TRAM) protein [8] (TRAP) since the protein, although not a signal sequence receptor, seems to be located at the translocation site. The a subunit of TRAP (TRAPa) can be crosslinked to a minor extent to various translocating chains, and the proportion of TRAPa among glycoprotein crosslinks appears to increase as chain length increases [8]. TRAPa is segregated to the rough portion of the ER [12], it can be crosslinked to membranebound ribosomes [13] and TRAP is associated in part with ribosomes after solubilization of rough microsomes with detergents [9]. The protein is a major constituent of the ER membrane of various species [lo]. Finally, antibodies against TRAPa and Fab fragments prepared from the antibodies inhibit the in vitro translocation of several secretory proteins [lo].The amino acid sequence of TRAPa was deduced from cloning of its cDNA and indicates that it is a single-spanning membrane protein [14]. Employing Nonidet P-40 as a detergent to solubilize canine pancreatic microsomes, it was found that TRAPa is tightly and stoichiometrically associated with a second protein, called TRAPP {previously SSRB) [6]. The cDNA of TRAPD has also been cloned and codes for a glycosylated, single-spanning membrane protein [6].In the present study, we have used digitonin as a milder detergent to solubilize the membranes a...

Receptor and non-receptor tyrosine kinases constitute a large family of proteins that play a pivotal role in hematopoiesis. Here we conducted a comprehensive survey of tyrosine kinase gene expression in primary erythroid progenitor cells from bone marrow by employing a PCR-based strategy that targets the conserved kinase encoding region. We demonstrate that erythroid progenitor cells express several receptor and nonreceptor tyrosine kinases, like c-kit, Jak1, Ryk, FAK, Syk, Arg, Csk and members of the insulin receptor family. Speci®c changes in the expression pro®le of tyrosine kinases were observed following di erentiation induction. We also report on the identi®cation of a new ligand dependent modulator of erythropoiesis, ®broblast growth factor receptor-4 (FGFR-4). FGFR-4 is e ectively expressed in erythroid progenitors and downregulated when cells di erentiate. Furthermore, the FGFR-4 ligand, basic ®broblast growth factor (bFGF), enhanced erythroid cell proliferation induced by SCF or insulin, and thus modulated both erythroid proliferation and di erentiation in vitro.

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