http://www.cs.duke.edu/courses/cps262/current/pdf/hafner.2010.pdf

Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP

https://rnajc.ucsf.edu/sites/rnajc.ucsf.edu/files/CastelloArticlePlusSupplementalJune2012.pdf

Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins

Post-transcriptional gene regulation (PTGR) concerns processes involved in the maturation, transport, stability and translation of coding and non-coding RNAs. RNA-binding proteins (RBPs) and ribonucleoproteins coordinate RNA processing and PTGR. The introduction of large-scale quantitative methods, such as next-generation sequencing and modern protein mass spectrometry, has renewed interest in the investigation of PTGR and the protein factors involved at a systems-biology level. Here, we present a census of 1,542 manually curated RBPs that we have analysed for their interactions with different classes of RNA, their evolutionary conservation, their abundance and their tissue-specific expression. Our analysis is a critical step towards the comprehensive characterization of proteins involved in human RNA metabolism.

/pdf/a-census-of-human-rna-binding-proteins-1mii7pfs5y.pdf

A census of human RNA-binding proteins.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2008.03.004

RNA-binding proteins and post-transcriptional gene regulation

The mRNA-Bound Proteome and Its Global Occupancy Profile on Protein-Coding Transcripts

Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) particles can be efficiently purified by a specific, rapid, and mild procedure using monoclonal antibodies to hnRNP proteins. We report here on the detailed analysis of the protein composition of immunopurified hnRNP particles from human HeLa cells. By two-dimensional gel electrophoresis, immunopurified hnRNP particles contain at least 24 polypeptides in the range of 34,000-120,000 daltons. The abundant 30,000-40,000 dalton proteins, A, B, and C, described previously, are a subset of these polypeptides. The protein compositions of hnRNP particles found in the nucleoplasm fraction and in the chromatin-nucleolar fraction are very similar. Upon addition of the polyanion heparin, most of the major proteins remain associated in heparin-resistant particles, and only several, mostly minor, proteins dissociate. This provides an aid in the classification of the proteins and an additional criterion for the definition of hnRNP particle components. Chromatography on single-stranded DNA (ssDNA)-agarose in a heparin- and moderate or high salt (higher than 300 mM NaCl)-resistant manner suggests that most, if not all, of these proteins are single-stranded nucleic acid-binding proteins. We describe a general method for the large-scale purification of hnRNP proteins by affinity chromatography on ssDNA columns and its use for the production of new monoclonal antibodies to hnRNP proteins.

/pdf/immunopurification-of-heterogeneous-nuclear-2entzmhwsi.pdf

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

Splicing in vitro of a messenger RNA (mRNA) precursor (pre-mRNA) is inhibited by a monoclonal antibody to the C proteins (anti-C) of the heterogeneous nuclear RNA (hnRNA)-ribonucleoprotein (hnRNP) particles. This antibody, 4F4, inhibits an early step of the reaction: cleavage at the 3' end of the upstream exon and the formation of the intron lariat. In contrast, boiled 4F4, or a different monoclonal antibody (designated 2B12) to the C proteins, or antibodies to other hnRNP proteins (120 and 68 kilodaltons) and nonimmune mouse antibodies have no inhibitory effect. The 4F4 antibody does not prevent the adenosine triphosphate-dependent formation of a 60S splicing complex (spliceosome). Furthermore, the 60S splicing complex contains C proteins, and it can be immunoprecipitated with 4F4. Depletion of C proteins from the splicing extract by immunoadsorption with either of the two monoclonal antibodies to the C proteins (4F4 or 2B12) results in the loss of splicing activity, whereas mock-depletion with nonimmune mouse antibodies bodies has no effect. A 60S splicing complex does not form in a C protein-depleted nuclear extract. These results indicate an essential role for proteins of the hnRNP complex in the splicing of mRNA precursors.

https://science.sciencemag.org/content/sci/231/4745/1534.full.pdf

Heterogeneous nuclear ribonucleoproteins: role in RNA splicing.

The packaging of heterogeneous nuclear RNA (hnRNA), the fate of hnRNA in the nucleus, and the conversion of hnRNA to mRNA are believed to occur as the hnRNA transcript is associated with specific proteins to form a ribonucleoprotein complex termed the hnRNP complex. The identity and organization of the protein constituents of the hnRNP have been a matter of considerable controversy. We report here the isolation of the hnRNP complex from vertebrate cell nuclei, employing immunoprecipitation with monoclonal antibodies against the major proteins that are in contact with hnRNA in HeLa cells. Rapid immunoprecipitation from HeLa nucleoplasm with two different monoclonal antibodies to the hnRNP C proteins (41 and 43 kDa) isolates a similar complex that contains proteins and hnRNA of up to approximately equal to 10 kilobases. The major steady-state [35S]methionine-labeled proteins of the isolated complex are of 34 kDa, 36 kDa (A1 and A2), 37 kDa, 38 kDa (B1 and B2), 41 kDa, 43 kDa (C1 and C2), and doublets at 68 kDa and at 120 kDa. Additional proteins from 45 kDa to very high molecular mass are also seen. The major proteins of the complex appear identical by NaDodSO4/polyacrylamide gel electrophoresis to genuine hnRNP proteins--those which become crosslinked by UV light to the hnRNA in vivo. Immunoprecipitation with a different, noncrossreacting monoclonal antibody to the 120-kDa protein isolates an apparently identical complex of proteins that are present at a similar relative stoichiometry. Similar hnRNP complexes are found in rodent and avian cells. Nuclease digestions indicate that RNA plays a role in maintaining the integrity of the structure and that intact RNA of approximately equal to 125 nucleotides is sufficient to hold the complex of proteins together. The coimmunoprecipitation of the hnRNA and of all of the proteins through antibodies against different genuine hnRNP proteins and from divergent species strongly suggests that the hnRNP complex is a unitary structure of consistent, defined, and conserved components.

https://www.pnas.org/content/pnas/81/23/7471.full.pdf

Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a unique supramolecular assembly.

Publisher Summary This chapter describes methods for RNA–protein cross-linking in living cells as a means of identification of RNA-binding proteins in vivo . The chapter focuses on heterogeneous nuclear RNA (hnRNA)- and messenger RNA (mRNA)-binding proteins. The most stringent definition of genuine ribonucleoproteins (RNPs) is that these are proteins, which are bound directly to the RNA of interest in the living cell. UV cross-linking of RNP complexes takes advantage of the fact that UV light of sufficient intensity generates highly reactive species of RNA, which react virtually indiscriminately with molecules, including proteins, with which the RNA is in stable, direct contact. The cross-linked proteins can be released from the RNA by exhaustive RNase digestion and can be analyzed by gel-electrophoretic techniques. This method provides a powerful tool for the identification of bona fide RNP proteins. The fractionation of cells into nuclear and cytoplasmic components prior to the chromatographic step allows the distinction between hnRNA-containing RNP complexes (hnRNPs), from the nuclear fraction, and mRNAcontaining RNP complexes (mRNPs), from the cytoplasmic fraction.

Ultraviolet-induced cross-linking of RNA to proteins in vivo.

The interaction of mRNA with proteins in vesicular stomatitis virus (VSV)-infected cells was studied by photochemical cross-linking in intact cells. The major [35S]methionine-labeled proteins which became cross-linked by UV light to mRNA in uninfected and in VSV-infected HeLa cells were similar and had apparent mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to 135, 93, 72, 68, 53, 50, 43, and 36 kilodaltons. The proteins which were cross-linked in vivo specifically to the five mRNAs of VSV were labeled through radioactive nucleotides incorporated only into VSV mRNAs under conditions (5 micrograms of actinomycin D per ml) in which only VSV mRNAs are labeled. The same major mRNP proteins that became cross-linked to host mRNAs also became cross-linked to VSV mRNAs, although several quantitative differences were detected. Photochemical cross-linking and immunoblotting of cross-linked mRNPs with VSV antiserum demonstrated that in addition to host proteins VSV mRNAs also became cross-linked to the VSV-encoded N protein. The poly(A) segment of both host and VSV mRNAs was associated in vivo selectively with the 72-kilodalton polypeptide. The major proteins of mRNA-ribonucleoprotein complexes are therefore ubiquitous and common to different mRNAs. Furthermore, since the major messenger ribonucleoproteins interact also with VSV mRNAs even though these mRNAs are transcribed in the cytoplasm, it appears that nuclear transcription and nucleocytoplasmic transport are not necessary for mRNA to interact with these proteins.

Interaction of mRNA with proteins in vesicular stomatitis virus-infected cells.

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