Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCβ. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete Mr ≃ 2 × 106 dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.

Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA

The protein component of ribonuclease P (RNase P) binds to the RNA subunit, forming a functional ribonucleoprotein complex in vivo and enhancing the affinity of the precursor tRNA (pre-tRNA) substrate. Photocrosslinking experiments with pre-tRNA bound to RNase P reconstituted with the protein component of Bacillus subtilis ribonuclease P (P protein) site specifically modified with a crosslinking reagent indicate that: (i) the central cleft of P protein directly interacts with the single-stranded 5′ leader sequence of pre-tRNA, and (ii) the orientation and register of the pre-tRNA leader sequence in the central cleft places the protein component in close proximity to the active site. This unique mode of interaction suggests that the catalytic active site in RNase P occurs near the interface of RNA and protein. In contrast to other ribonucleoprotein complexes where the protein mainly stabilizes the active tertiary fold of the RNA, a critical function of the protein component of RNase P is to alter substrate specificity and enhance catalytic efficiency.

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed.

Structural Requirements of Tom40 for Assembly into Preexisting TOM Complexes of Mitochondria

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.