Independent deposition of heterogeneous nuclear ribonucleoproteins and small nuclear ribonucleoprotein particles at sites of transcription.

Abstract

The major nuclear ribonucleoproteins (RNPs) involved in pre-mRNA processing are classified in broad terms either as small nuclear RNPs (snRNPs), which are major participants in the splicing reaction, or heterogeneous nuclear RNPs (hnRNPs), which traditionally have been thought to function in general pre-mRNA packaging. We obtained antibodies that recognize these two classes of RNP in Drosophila melanogaster. Using a sequential immunostaining technique to compare directly the distribution of these RNPs on Drosophila polytene chromosomes, we found that the two patterns were very similar qualitatively but not quantitatively, arguing for the independent deposition of the two RNP types and supporting a role for hnRNP proteins, but not snRNPs, in general transcript packaging.Both heterogeneous nuclear ribonucleoproteins (hnRNPs; reviewed in refs. 1 and 2) and small nuclear ribonucleoproteins (snRNPs; reviewed in ref.3) are deposited cotranscriptionally on eukaryotic RNA polymerase II transcripts (4-8). Whereas the major basic hnRNP proteins have been considered traditionally to function in general pre-mRNA packaging (2, 9), they have been proposed recently to be specific splicing cofactors or to be preferentially associated with splice junction sequences (10-15). snRNPs are major participants in the splicing reaction (3) but have been implicated recently in general packaging as part of a previously assembled unitary processing complex also containing hnRNPs (5, 6). The various proposals predict different amounts and ratios of the two protein types on nuclear pre-mRNA molecules at chromosomal sites of transcription, which is the issue we have addressed by sequential immunostaining.The core hnRNP proteins (A, B, and C proteins of 32-45 kDa) were originally identified as the major proteins that are associated with newly synthesized pre-mRNA (in the form of 30-50S RNP particles) when it is extracted from nuclei (reviewed in refs. 1 and 2). This observation, together with their nuclear abundance, their ability to bind single-stranded nucleic acids regardless of sequence, and their helixdestabilizing properties, led to the notion that these core hnRNP proteins are involved in general pre-mRNA packaging, much as the histones are involved in the general packaging of DNA (1, 2). However, more recent investigations of hnRNP proteins, using in vitro splicing or in vitro RNA binding studies, have suggested that these proteins play a role in the splicing reaction (10-12), that they bind with high affinity to sequences at 3' splice sites (13,14), and that they are dependent on snRNPs for acquisition of a crosslinkable association with RNA (13). These in vitro studies have led to a reappraisal of the independent structural role of hnRNP proteins in pre-mRNA packaging towards a view that they are a few of the many required cofactors for splicing. The simplest version of this view would predict a constant stoichiometry of snRNPs and the core hnRNP proteins on pre-mRNA, in amounts that correlate with the number of splicing sign...

The Drosophila sex determination gene snf encodes a nuclear protein with sequence and functional similarity to the mammalian U1A snRNP protein.

“…6D). These results are consistent with those obtained previously by Amero et al {1992) who found that mAb 4G3 detected an antigen that is associated with many sites throughout the polytene chromosomes. As might be expected, we found that the staining of chromosomes is not limited to the salivary gland.…”

“…Previously, a monoclonal antibody (mAb 4G3} directed against the human U2B" was shown to detect a Drosophila nuclear antigen that is similar in size to the predicted 28-kD SNF protein (Habets et al 1989;Amero et al 1992;Bopp et al 1993). Is this antigen SNF?…”

Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: Focus on hnRNP E1's multifunctional regulatory roles

“…Evidence against a sole hnRNP complex, and for differential association of hnRNPs with pre-mRNAs, was obtained from both in vitro RNAbinding studies and immunohistochemical studies. The idea of transcript-specific packaging is further supported by the finding that the ratio of individual hnRNPs varies with different primary transcripts (Amero et al 1992;Matunis et al 1993). …”

Distribution of pre-mRNA splicing factors at sites of RNA polymerase II transcription.

“…Importantly, a range of immunostaining intensities relative to anti-Pol II was observed in both studies, raising the possibility that these two SR proteins accumulate differently on nascent transcripts in this experimental system. Other classes of pre-mRNA processing factors, namely the heterogenous nuclear RNPs (hnRNPs) and snRNPs, have been shown to associate differentially with nascent transcripts on polytene chromosomes and amphibian lampbrush chromosomes (Amero et al 1992;Matunis et al 1993;Wu et al 1993), but the functional significance of these differences is unknown.…”

“…This has led to the suggestion that speckles contain the highest concentrations of splicing factors--perhaps serving as storage or assembly sites--and that lower concentrations may be present where splicing occurs (Fakan et al 1984;Fakan 1994;Xing et al 1995;Huang and Spector 1996). Interestingly, SR proteins and splicing snRNPs are detected at active sites of transcription on polytene and lampbrush chromosomes, which are particularly well-suited for cytological observation (Roth et al 1989(Roth et al , 1991Champlin et al 1991;Amero et al 1992;Kim et al 1992;Wu et al 1993;Bauren et al 1996). Although splicing factors accumulate at very large and highly active viral transcription units in infected mammalian cells (cf.…”