GoldCLIP: Gel-omitted Ligation-dependent CLIP.

Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein-RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein (PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.

Isolation of a sequence-specific RNA binding protein, polypyrimidine tract binding protein, using RNA affinity chromatography.

Many important cellular processes are mediated by sequence-specific RNA binding proteins, and it is often necessary to purify these proteins. When the RNA binding site is known, it is convenient to use this RNA as a matrix for affinity purification. The intronic splicing silencer (ISS) element present upstream of the N1 exon of the c-src pre-mRNA is a high-affinity binding site for the polypyrimidine tract binding protein (PTB). Using a 5'-biotinylated ISS RNA and PTB as an example, I describe a one-step method for affinity chromatography of RNA binding proteins from nuclear extracts.

Expression pattern of three putative RNA-binding proteins during early development of the sea urchin Paracentrotus lividus.

We report the expression patterns of three transcripts encoding RNA-binding proteins during early development of the Mediterranean sea urchin Paracentrotus lividus. Two of these genes encode KH-domains RNA-binding proteins closely related to the vertebrate neuro-oncological ventral antigen 1 (Nova) and RING Finger and KH-domain (RKHD). The third encodes the sea urchin ortholog of the polypyrimidine tract binding protein (PTB). Zygotic expression of nova and rkhd starts at mesenchyme blastula stage and is restricted to the presumptive endoderm territory. During gastrulation, expression of nova is restricted to the midgut and hindgut, while expression of rkhd become more complex and includes the foregut and hindgut territories as well as previously unknown territories within the ectoderm. PTB is first expressed ubiquitously but starting at the late gastrula stage, then PTB transcripts become highly enriched in the foregut and oral ectoderm. We further report that expression of nova and rkhd in the endomesoderm is under the control of the Wnt/beta-catenin pathway and occurs in a cell-autonomous manner while expression of rkhd and PTB in the oral ectoderm is regulated by Nodal signaling.

Micro-scale open-tube capillary separations of functional proteins.

This article describes a novel technique whereby fully functional proteins or multiprotein complexes are efficiently extracted from biological samples to chemically derivatized walls of fused-silica open-tube capillary columns. Proteins are eluted with very high yields into elution volumes that are smaller in volume than the internal volume of the open-tube capillary column itself, thereby achieving 100-fold increases in target protein concentrations from starting samples of less than 1 ml. The open-tube capillary columns are designed for single use; combined with the physical and chemical characteristics of the open-tube capillary column, this provides exceptional purity to the eluted proteins. Affinity-based open-tube capillary columns are demonstrated here to purify, enrich, and maintain functionality for a monomeric and dimeric enzyme, a low-abundance HeLa nuclear complex, and a light-harvesting octadecameric membrane protein complex. The design of the open-tube capillary column allows for facile direction of the processed protein sample to any number of final detection techniques and is capable of generating final protein concentrations required for many structural biology experiments. The open-tube capillary columns are also characterized by exceptional ease of use. Current designs allow for up to 10 open-tube capillary columns to be applied simultaneously with no fundamental impediments to even greater parallel operation.

Polypyrimidine tract-binding protein interacts with the 3' stem-loop region of Japanese encephalitis virus negative-strand RNA.

The 3' stem-loop (SL) region of positive- and negative-strand RNA of Japanese encephalitis virus (JEV), like that of other flaviviruses, may function as cis-acting signals during RNA replication. In order to demonstrate the specific interaction between JEV 3' SL regions and BHK-21 cellular proteins, we performed gel mobility shift assay and UV-induced cross-linking assay. We identified seven cellular proteins of 110, 87, 67, 45, 38, 34, and 30 kDa that bound to the (+)3' SL RNA, and eight cellular proteins of 138, 110, 87, 67, 55, 52, 38, and 34 kDa that bound to the (-)3' SL RNA. The 55 kDa protein was identified as the polypyrimidine tract-binding (PTB) protein by immunoprecipitation assay. These data suggest that the 3' SL regions of JEV-RNA of both polarities may act as recruiting signals for the components of viral replication complexes including host cell-derived PTB protein.

Two cellular proteins that interact with a stem loop in the simian hemorrhagic fever virus 3'(+)NCR RNA.

Both full-length and subgenomic negative-strand RNAs are initiated at the 3' terminus of the positive-strand genomic RNA of the arterivirus, simian hemorrhagic fever virus (SHFV). The SHFV 3'(+) non-coding region (NCR) is 76 nts in length and forms a stem loop (SL) structure that was confirmed by ribonuclease structure probing. Two cell proteins, p56 and p42, bound specifically to a probe consisting of the SHFV 3'(+)NCR RNA. The 3'(+)NCR RNAs of two additional members of the arterivirus genus specifically interacted with two cell proteins of the same size. p56 was identified as polypyrimidine tract-binding protein (PTB) and p42 was identified as fructose bisphosphate aldolase A. PTB binding sites were mapped to a terminal loop and to a bulged region of the SHFV 3'SL structure. Deletion of either of the PTB binding sites in the viral RNA significantly reduced PTB binding activity, suggesting that both sites are required for efficient binding of this protein. Changes in the top portion of the SHFV 3'SL structure eliminated aldolase binding, suggesting that the binding site for this protein is located near the top of the SL. These cell proteins may play roles in regulating the functions of the genomic 3' NCR.