5.6 The nucleolus and nuclear import and export
The messenger RNA is not the only RNA family for which the nucleus is responsible. Ribosomal RNA transcription and processing, and ribosome assembly are crucial aspects of nuclear function. Indeed the site of these activities is the most prominent morphological feature within the nucleus; it’s been known for many years as a body called the nucleolus. Genes specifying ribosomal sequences are transcribed into pre-ribosomal-RNAs (pre-rRNAs). Virtually all cellular RNAs undergo post-transcriptional processing and modification. Pseudouridylation (which is the conversion of uridine to pseudouridine at specific sites in an RNA sequence) and 2’-O-methylation of the ribose sugar are the most common internal modifications. They are applied to three major types of stable RNA: the spliceosomal snRNAs, ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). As is the case for pre-mRNA processing, pre-rRNA processing is done by families of proteins working with numerous small nucleolar RNAs (snoRNAs) in the form of RNP complexes (snoRNPs) that base pair with specific parts of the pre-rRNA sequence to carry out their modification.
The snoRNAs are themselves produced from precursor transcripts and have to be targeted to the nucleolus so that they can guide the critical modifications of the rRNA. As we’ve indicated above, the snoRNPs can also modify spliceosomal snRNAs and as this could affect the way pre-mRNAs are processed it is evident that the nucleolus is responsible for more than just ribosome assembly. Nevertheless, the nucleolus where ribosomal subunits are manufactured and the proteins needed to assemble ribosomes are synthesised in the cytoplasm, like all proteins, so they must be imported into the nucleus and also targeted to the nucleolus. Subsequently, ribosomal subunits need to be re-exported to the cytoplasm. Ribosomal subunits are large macromolecular assemblies and the way they are translocated to the cytoplasm is not yet known. More is known about export of mRNA from the nucleus.
The nucleus is separated from the cytoplasm by a double membrane called the nuclear envelope, which isolates and protects the DNA from the turmoil of the cytoplasm (in prokaryotes the processes that depend on DNA take place in the cytoplasm). In eukaryotes many proteins and RNAs are transported across the nuclear envelope. This is known as nuclear–cytoplasmic trafficking and features both import into and export from the nucleus. It occurs through the nuclear pore complexes (NPCs), which are complex assemblies that are embedded in the double-membrane of the nuclear envelope. NPCs provide channels (about 9 nm in diameter) that allow passage of ions and small molecules (less than about 50 kDa) by diffusion, but proteins, RNAs, and ribonucleoprotein (RNP) particles larger than 9 nm are selectively transported through NPCs by an energy-dependent mechanism. The trafficking is selectively regulated by developmental and environmental signals.
The overall three-dimensional architecture of the NPC is conserved from yeast to higher eukaryotes though the yeast NPC is smaller than those found in vertebrates. The protein components of NPCs are called nucleoporins; the yeast NPC is composed of 30-50 different nucleoporins (up to 100 in vertebrates). We are just beginning to understand the three-dimensional molecular architecture of the NPC but, crudely, it looks rather like the ball valve of a swimmer’s snorkel. There is a ring of nucleoporins on the cytoplasmic side of the pore that extends through the nuclear membrane, matched with another ring on the nuclear side of the pore. Between the rings there is a central plug (also called the transporter because some specimens seem to have cargo trapped within), and eight short fibrils extend from the cytoplasmic ring into the cytoplasm whereas the nuclear ring anchors a basket made from eight long thin filaments (Stoffler et al., 1999).
How this highly organised tunnel actually works remains a mystery, but the transport process itself depends on a family of soluble transporter proteins known as importins or exportins (both also called karyopherins) that carry proteins and RNA between the cytoplasm and nucleus. Proteins or RNAs ready for transport (the cargo) contain specific sequences that identify them. The nuclear localisation signal (NLS) is recognised by importins, and the nuclear export signal (NES) sequence is recognised by exportins. Transporter and cargo then dock at the mouth of the NPC and deliver the transport cargo. Importantly, for large RNA molecules such as mRNA - which is translocated as a RNP - the signals for export are not the mRNA itself but on the proteins, mostly hnRNP and splicing factors, that have guided the transcript through final processing. This emphasises again the integration and co-operation of the machineries that function in gene expression.
Interaction of cargo with transporter is modulated by the small protein Ran, which is a GTPase. GTP hydrolysis by Ran does not provide the energy for transport but regulates the assembly and disassembly of transport complexes. Effectively, the nucleotide bound state of Ran identifies the compartment: Ran-GTP in the nucleus, Ran-GDP in the cytoplasm. For importins, the binding of cargo and Ran-GTP is antagonistic (so importins drop their cargo in the nucleus) and for exportins the binding of cargo and Ran-GTP is co-operative (so exportins are loaded in the nucleus). Of course, it’s not as simple as that; there are several accessory factors involved in maintaining Ran in its proper GTP or GDP bound state, including a nuclear nucleotide exchange factor (RCC1), a cytoplasmic Ran-GTPase activating protein (RanGAP), a protein called RanBP1, and nuclear transport factor 2 (NTF2). Though the significance of this is uncertain, there are multiple signals and multiple pathways for both nuclear import and export; 14 members of the importin/exportin family can be predicted from the genomic sequence of Saccharomyces cerevisiae, fewer, so far, have been identified in higher eukaryotes (Adam, 1999).
By this stage in our description we’ve reached the point of exporting the gene transcript(s) to the cytoplasm. We’ll continue our story of what subsequently happens to the ribosomal subunits and mRNAs in the cytoplasm later [CLICK HERE to see it now if you just can't wait]. At the moment we want to proceed to the other prime function of the nucleus, namely storage and transmission of the cell’s genetic content.
Updated January 18, 2017