Assembly and transport of messenger and ribosomal RNP particles in the dipteran Chironomus tentans

Gene expression in eukaryotic organisms commences with the synthesis of a precursor RNA, which has to be extensively processed to yield a mature RNA molecule. In addition to processing, the RNA molecule needs to associate with a proper set of proteins. Some of these proteins mediate structure, some are involved in the maturation and export processes, and others might not have a function before entering the cytoplasm. The significance of a correct protein composition of the ribonucleoprotein (RNP) particle remains to be explored. The RNP particle requires transport from the site of transcription, through the nucleoplasm and the nuclear pore complex (NPC), to the cytoplasm, where messenger RNAs (mRNAs) are translated into protein and ribosomal RNPs (rRNPs) form mature ribosomes. The NPC and its components are likely to be involved in the regulation of RNA export by interacting specifically with export substrates.

We have used the unique properties of the larvae of the dipteran Chironomus tentans to study the relation of four proteins to the biogenesis of mRNA in situ. The salivary gland cells contain polytene chromosomes with the transcriptionally active regions blown up as puffs. A few giant puffs, called Balbiani rings (BRs), generate large RNP particles that can be visualised in the electron microscope (EM) during assembly on the gene and transport to and through the NPCs.

We have found that a putative translational regulator, Ct-p40/50, was loaded onto nascent BR pre-mRNA transcripts already during transcription. In addition, it was present in many other chromosomal loci, suggesting that Ct-p40/50 is a general RNA binding protein. Ct-p40/50 also appeared in the nucleoplasm and was abundant in the cytoplasm. In both compartments, it was bound to poly(A)+ RNA. Ct-p40/50 accompanied the BR transcript all the way through the nucleoplasm to the nuclear pores and into the cytoplasm, where it was mainly present in polysomes. We conclude that the cotranscriptional loading of a putative translational regulator represents an early programming of the cytoplasmic fate of mRNA.

The nuclear poly(A) binding protein PABPN1, known to regulate the length of the poly(A) tail was found to be associated with RNA polymerase (pol) II along the chromatin axis of the BR gene. After transcription, PABPN1 associated with the transcript and accompanied the BR mRNP to the NPC. A small fraction of the protein was found in the cytoplasm, suggesting that PABPN1 is displaced from mRNPs during or shortly after passage through the NPC. Our results suggest that the assembly of PABPN1 onto the poly(A) tail is coupled to transcription.

We have characterised a protein that specifically recognises one of the transcriptional BR puffs, the BR3. The protein was designated Ct-hrp130 and showed a sequence organisation strikingly similar to the human transcriptional regulator CA150. The BR3 gene is especially rich in introns, and we suggest that Ct-hrp130, like CA150, can down-regulate transcription. This would enable splicing of the BR3 gene to occur cotranscriptionally.

Injection of an antibody to the NPC component Nup153 caused accumulation of both mRNA and rRNA in the nucleoplasm. Analysis by EM revealed that the mRNP particles bound to the top of the basket structure, while particles already engaged in translocation through the NPC completed their passage. This demonstrated that the block occurred at a stage between the binding of the export substrate to the NPC and its translocation through the pore. The fact that rRNA and mRNA accumulated in the nucleoplasm and that no RNPs lined up in front of the pore argues for a random movement of export substrates to the pore. We suggest that Nup153 is directly involved in the translocation process of mRNA and rRNA through the NPC at a stage right before the cargo enters the basket of the NPC.

In summary, we have studied specific aspects of the assembly and transport of messenger and ribosomal RNP particles. The PABPN1 protein was present already on the RNA polymerase II, suggesting that it can be transferred from the polymerase onto the poly(A) tail of the transcript concomitant with transcription. At certain genes, the Ct-hrp130 protein might slow down transcription of intron-rich transcripts to enable splicing to take place cotranscriptionally. When an antibody to Nup153 was injected into nuclei, export of messenger and ribosomal RNA was specifically blocked at a stage before the entry of the RNPs into the basket of the NPC, indicating that Nup153 is acting at the top of the basket. The cotranscriptional loading of certain proteins, like the putative translational regulator Ct-p40/50, onto nascent RNA suggests an early programming of not only the nuclear but also the cytoplasmic fate of the mRNA.

List of scientific papers

I. Soop T, Nashchekin D, Zhao J, Sun X, Alzhanova-Ericsson AT, Bjorkroth B, Ovchinnikov L, Daneholt B (2003). A p50-like Y-box protein with a putative translational role becomes associated with pre-mRNA concomitant with transcription. J Cell Sci. 116(Pt 8): 1493-503.
https://pubmed.ncbi.nlm.nih.gov/12640034

II. Bear DG, Fomproix N, Soop T, Bjorkroth B, Masich S, Daneholt B (2003). Nuclear poly(A)-binding protein PABPN1 is associated with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore. Exp Cell Res. 286(2): 332-44.
https://pubmed.ncbi.nlm.nih.gov/12749861

III. Sun X, Zhao J, Kylberg K, Soop T, Palka K, Aissouni Y, Sonnhammer E, Visa N, Alzhanova-Ericsson A, Daneholt B (2003). Identification and characterization of a transcript-specific hnRNP protein in the salivary glands of Chironomus tentans. [Manuscript]

IV. Soop T, Ivarsson B, Bjorkroth B, Cordes V, Daneholt B (2003). Nup153 affects the entrance of messenger and ribosomal RNPs into the nuclear basket during export. [Manuscript]