We have dissected the nucleocytoplasmic shuttling pathway of hnRNP A1 into at least three distinct classes of RNPs with characteristics of sequential stages in mRNA formation. Among these, we identified a likely intermediate nuclear mRNP (nmRNP) complex (or set of complexes) that contains mRNA with associated shuttling hnRNP proteins but no pre-mRNA or nonshuttling hnRNP proteins. The strategy for the isolation of these complexes is based on the different nucleocytoplasmic traffic characteristics of hnRNP proteins and exploits differences in subcellular associations of RNP complexes at different stages of maturation in combination with specific immunopurification of RNPs. The immunopurification approach taken here has been used successfully in the past to isolate hnRNP complexes under conditions that minimize disruption and rearrangements of the complex (13
). Therefore, the complexes described in this study are likely to represent endogenous RNPs that were assembled in vivo.
The three classes of RNP complexes described here can be distinguished from each other by their RNA and protein compositions and by their association with different subcellular fractions. One class corresponds to the previously described nucleoplasmic hnRNP complexes (13
), which contain shuttling and nonshuttling hnRNP proteins and which we show here contain both pre-mRNA and mRNA. This is distinct from a second class of RNPs, also associated with nuclei, which display characteristics of mature nuclear mRNPs (nmRNPs). Specifically, they contain mRNA but no detectable pre-mRNA. They also contain shuttling hnRNP proteins as well as the nuclear mRNA export factor REF, as is the case with hnRNP complexes, but no nucleus-retained hnRNPs such as C1/C2 and U. In addition, there are several proteins specifically associated with nmRNPs that are not found in hnRNP complexes. RNP complexes associated with hnRNP A1 are also found in the cytosol, where A1 is associated with mRNA as well as with the major cytoplasmic mRNP protein PABP1 (19
) and with transportin, the nuclear transport receptor for hnRNP A1 (62
). Importantly, all three types of complexes contain proteins in common, as well specific proteins that are not found in the other complexes.
The existence of distinct RNP complexes of hnRNP proteins was suggested by a number of previous studies, which indicated that nuclear formation of mRNAs from pre-mRNAs, as well as nucleocytoplasmic transport of mRNAs, is accompanied by substantial changes in the proteins associated with these RNAs (see the introduction). In addition, the major proteins bound to mRNA in the cytoplasm at steady-state levels are different from those associated with pre-mRNA in the nucleus (16
), indicating a wholesale exchange of mRNP for hnRNP proteins as the mRNA is exported from the nucleus to the cytoplasm. Taking these previous observations together with our findings, the distinct RNP complexes that we have isolated can best be fit into a temporal sequence of events in which the hnRNP complexes represent the initial pre-mRNPs as well as early postsplicing mRNPs in which RNA polymerase II-transcribed transcripts are found (17
). In agreement with this, pre-mRNA and mRNA for both β-actin and TAFII30 are detected in these complexes (Fig. ). In addition, the mRNA export factor REF is also present in hnRNP complexes. The presence of spliced mRNA in hnRNP complexes indicates that many of the subsequent changes in RNP protein composition represented by the additional mRNPs described here occur after processing of pre-mRNA to mRNA.
In contrast to hnRNPs, the fractionation properties of the cytosolic complexes and their association with transportin and PABP1 (a primarily cytosolic mRNP protein [19
]) indicate that they represent the last stage(s) in the nucleocytoplasmic shuttling of hnRNP A1 during its transit in the cytoplasm. These results also indicate that PABP1 can bind mRNA prior to complete release of hnRNP A1 from the same mRNA. By contrast, no significant amounts of PABP1 copurify with hnRNP K in the cytosolic fraction (Fig. B). This suggests that hnRNP K dissociates from mRNA prior to binding of PABP1 and therefore prior to release of hnRNP A1. Indeed, electron microscopy studies of BR mRNP export in C. tentans
have also shown different proteins dissociating from the mRNP in the cytoplasm at different stages following its nuclear export (for a review, see reference 15
). We cannot determine from these results whether both transportin and PABP1 coexist simultaneously in the same complexes with hnRNP A1 or whether they interact with hnRNP A1 in distinct complexes. The specificity of the interaction of hnRNP A1 with transportin is underscored by the absence of transportin in association with hnRNP K/J (Fig. B), in agreement with previous findings that hnRNP K does not require transportin for its nuclear import (47
). It is noteworthy that the number of proteins that can be cross-linked to mRNA in the cytoplasm is substantially larger than the number of proteins associated with hnRNP A1 (16
) (Fig. ). This would be consistent with a transient nature of the cytosolic hnRNP A1-containing mRNPs (supported by immunofluorescence microscopy data [60
]), indicating that additional proteins associate with cytoplasmic mRNA once hnRNP A1 is released from the complex (and therefore such proteins would not be coimmunoprecipitated with hnRNP A1).
The third class of RNP complexes, which are associated with hnRNP A1 in the Triton-extracted fraction, is of particular interest because their properties are consistent with those hypothesized for nuclear mRNPs at late stages of mRNA formation and possibly as substrates for nuclear export of mRNA. This conclusion is supported both by their protein and RNA compositions, as described above, and by their subcellular fractionation properties. Specifically, these nmRNPs are associated with the nuclear fraction and are not solubilized with digitonin under conditions that retain the integrity of the nuclear envelope (1
). Therefore, they are unlikely to represent soluble cytoplasmic complexes. On the other hand, they are readily released by Triton X-100 treatment of the digitonin-insoluble fraction, which contains nuclei as well as insoluble cytoplasmic structures. Treatment of nuclei with nonionic detergents is known to result in selective release of some nuclear contents, including RNA export factors, and we have observed this to also be the case for the nucleoplasmic pool of another abundant nuclear protein, nucleolin, with its associated rRNA (4
; Triolo and Piñol-Roma, unpublished data). It is unlikely that the complexes released by treatment with Triton X-100 originate from an otherwise insoluble cytoplasmic pool of mRNPs associated with the cytoskeleton. It has been reported that translated mRNA associates with the cytoskeleton, and there are conflicting reports as to the sensitivity of cytoskeleton-associated mRNA to treatment with detergents (6
). While we have not completely ruled out a cytoplasmic origin of these complexes, we consider this unlikely since they are not released into the soluble cytosolic fraction by treatment of cells with a variety of cytoskeleton-disrupting conditions (S. Mili and S. Piñol-Roma, unpublished observations). The properties of these RNPs, therefore, indicate that they are precursors to the cytosolic mRNPs described here and that they correspond to a later stage in mRNA formation than (and are a product of) the pre-mRNA-containing hnRNP complexes. This suggests strongly that these nmRNP complexes are a novel intermediate in the pathway of mRNA formation.
An important finding presented here is that several of the proteins in the nmRNPs are specific to these complexes and are not found in hnRNP complexes. The most prominent among them is LRP130, to which no specific function had been attributed (24
) and which our results indicate binds specifically to mRNA. Importantly, a protein with electrophoretic mobility similar to that of LRP130 is associated with all shuttling hnRNP proteins that we have examined thus far, including hnRNP A1 and hnRNP K/J (this work) and hnRNP A2/B1/B2 (Mili and Piñol-Roma, unpublished observations). Analysis of the LRP130 amino acid sequence revealed no readily apparent RNA-binding motifs. However, we show here that it binds poly(A)+
RNA in vivo, as it is readily cross-linked to mRNA by UV irradiation of living cells (Fig. ). The only recognizable amino acid sequence motif in LRP130 is the recently described PPR motif (68
). Other proteins with PPR motifs have also been shown to bind RNA and/or participate in mRNA metabolism, raising the possibility that the PPR motif itself is an RNA-binding motif (68
). Therefore, LRP130 is an RNA-binding protein with none of the known RNA-binding motifs, suggesting that it contains a novel type of RNA-binding domain. A recent study with Drosophila melanogaster
has shown that BSF, a protein highly similar to LRP130, has RNA-binding activity and is involved in regulating the stability of bicoid
Other proteins associated with this intermediate and not present in nucleoplasmic hnRNP complexes are the D0 and E0 proteins. There are four alternatively spliced isoforms of hnRNP D: D2 (p45), D1 (p42), D02 (p40), and D01 (p37) (74
). Of these, D2 and D1 isoforms are preferentially associated with hnRNP complexes (Fig. B). D1 and D2 contain a specific amino acid sequence that is encoded by an alternatively spliced exon that is absent from the D01 and D02 isoforms. By contrast to D1 and D2, D01 and D02 are specifically associated with nmRNPs. While all D protein isoforms are predominantly nuclear at steady-state levels, they differ in their ability to shuttle between the nucleus and the cytoplasm. Interestingly, the amino acid sequence specific to the D1/D2 isoforms has been proposed to be responsible for retaining them in the nucleus by mediating their interaction with the nuclear matrix-associated factor SAF-B. D01 and D02, which lack this sequence, do not associate with SAF-B and are able to shuttle (3
). These properties are consistent with our observation that D1- and D2-containing hnRNP complexes are resistant to extraction with nonionic detergent, whereas D01- and D02-containing nmRNP complexes seem to be relatively freely diffusible in the nucleoplasm, since they are readily extracted by mild detergent treatment. This distribution of hnRNP D protein isoforms is reminiscent of the Drosophila
RNA-binding protein How, which exists in two isoforms with different subcellular distributions. It was proposed that the shorter isoform can compete directly with the longer nuclear isoform for binding to target RNAs, thereby releasing inhibition of nuclear export in a developmentally regulated manner (48
). We speculate that a similar mechanism could operate in the case of the hnRNP D proteins, with the D01 and D02 isoforms possibly displacing the nucleus-retained D1 and D2 isoforms at a specific stage prior to nuclear RNA export. The specific relationship between E1 and E0 is not known, but their immunological relatedness, together with precedent from other hnRNP proteins (9
), suggests that they may also be produced by alternative splicing from the same pre-mRNAs.
It is noteworthy that recruitment of a number of RNA-binding proteins onto mRNA in the nucleus has been shown or hypothesized to be required for subsequent function of these proteins in the cytoplasm, e.g., in RNA localization, stability, and regulation of translation (see, e.g., references 7
, and 40
). For example, the D. melanogaster
hrp40 protein, which is similar to hnRNP D, mediates cytoplasmic localization of a number of pair-rule transcripts, and this function requires recruitment of Hrp40 to the mRNA in the nucleus (34
). Furthermore, contributions of hrp40 to regulation of Gurken localization during oogenesis vary among specific hrp40 isoforms (52
). In vertebrate cells, recruitment of specific hnRNP D protein isoforms (also known as AUF1) to mRNA in the nucleus has been proposed to determine the subsequent function of these proteins in cytoplasmic regulation of mRNA stability (40
). These observations, therefore, are in agreement with our finding that specific isoforms of hnRNP proteins initiate their association with the mRNA in nuclear mRNP complexes. RNP complexes containing β-actin and TAFII30 pre-mRNA and mRNA exhibit similar fractionation properties, suggesting that the overall characteristics of the observed complexes are a general feature of most transcripts in the cell. However, it is likely that the actual relative amounts of different proteins in the RNPs vary among specific transcripts (43
), depending on the sequence characteristics and ultimate fate of the mRNA.
Based on these findings, we propose the model shown in Fig. for remodeling of pre-mRNP and mRNP complexes during nuclear maturation and export of mRNA. As previously shown, most hnRNP proteins associate with nascent transcripts produced by RNA polymerase II, in pre-mRNA-containing hnRNP complexes. In addition to hnRNP proteins, other components of the pre-mRNA processing machinery (including snRNPs) associate transiently with hnRNP complexes as the reactions leading to formation of mature mRNA take place. For transcripts derived from intron-containing genes, the RNAs are retained in the nucleus as long as spliceosomes can be formed on these RNAs (12
). In addition, RNA-bound proteins with NRSs such as hnRNP C1/C2 and D1/D2 may also mediate nuclear retention of the bound RNAs (50
). Completion of splicing leads to recruitment of a subset of mRNP-specific proteins, such as Y14, DEK, SRm160, and the mRNA export factor REF. Subsequent removal of NRS-containing proteins would release this mRNP from a “nuclear anchor,” possibly rendering the mRNP freely diffusible in the nucleoplasm. The mechanism for removal of NRS-containing proteins is not known. By analogy to the D. melanogaster
RNA-binding protein How (48
), this dissociation could result from displacement by alternatively spliced isoforms (as could be the case for hnRNP D and E) or by a different mechanism (e.g., through the action of helicases [29
]). This complex would also acquire additional mRNA-specific proteins such as LRP130 and hnRNP D01/D02, leading to formation of an nmRNP intermediate that is subsequently exported to the cytoplasm. Following export from the nucleus, shuttling hnRNP proteins remain associated in a transient cytosolic mRNP (cmRNP*). An exchange of cytoplasmic mRNP proteins for nmRNP proteins takes place, ultimately resulting in a distinct cytoplasmic mRNP (cmRNP) that serves as a substrate for cytoplasmic mRNA metabolism. Shuttling hnRNP proteins dissociate from the mRNA at different stages and are reimported into the nucleus. Therefore, RNP remodeling during mRNA maturation and export would involve stage-specific release of bound proteins, as well as stage-specific recruitment of additional RNA-binding proteins. Any of the stages depicted in this model are potential targets for regulation.
Model for the sequential changes in protein composition of complexes associated with pre-mRNA and mRNA during maturation and nuclear export of mRNA. See text for details.
At least two of the proteins in this nmRNP, namely, hnRNP A1 and hnRNP K, contain sequences that can mediate nuclear protein export (46
), and thus multiple NESs are found on individual mRNAs. Our results show that at least one of the mRNA export factors identified thus far in vertebrate cells, REF (71
), is also associated with the nmRNP. We have been unable to determine unambiguously whether TAP is also associated with the nmRNP. It is possible that the interaction of TAP with the nmRNP is too unstable or weak to survive the RNP isolation procedure, as suggested also by results from other laboratories (see, e.g., reference 77
). Experiments are now in progress to determine which (if any) additional known RNA export factors and/or NPC components, as well as other proteins recruited to mRNA through splicing (see the introduction), are present in or interact with this nmRNP. Additional nmRNP components may include members of the SR family of splicing factors, some of which also shuttle between the nucleus and the cytoplasm (10
). Experiments are also in progress to determine whether mRNAs derived from intronless genes also associate in similar complexes and with similar proteins.