Nuclear Speckle
Nuclear speckles ( English as "Kernsprenkel"), even Interchromatin granule cluster (ICG) called areas in are the nucleus , with splicing factors are enriched. They are located in the interchromatin regions of the nucleoplasm of mammalian cells. When viewed with an immunofluorescence microscope, they usually appear as irregularly shaped structures of different sizes in the cell nucleus. There are 20 to 50 speckles per nucleus. These objects are dynamic structures: their components can continuously exchange with the nucleoplasm and other core areas, including active transcription sites.
SnRNPs and other splicing factors accumulate in them in high local concentrations . The presence in the speckles of many other factors involved in mRNA production by RNA polymerase II supports their close relationship with gene expression . Although most speckles do not appear to contain DNA , highly expressed genes can still be found associated with speckles. This agrees with an important role of the speckles in coordinating the provision and / or recycling of factors for transcription and mRNA processing.
Studies of the composition, structure, and dynamics of speckles have provided an important paradigm for understanding the functional organization of the core and the dynamics of the gene expression machine.
history
Nuclear speckles were first recognized as storage and modification sites for splicing factors. Further studies on transcription and mRNA maturation and export showed a more general role of speckles in splicing in RNA metabolism.
The first detailed description of Nuclear Speckles was made in 1910 by Santiago Ramón y Cajal . Ramón y Cajal used acidic aniline stains to identify structures that he called "grumos hialinas". The term "speckle" was first coined in 1961 by J. Swanson Beck after examining sections of rat liver. Two years earlier, these spots were identified by Hewson Swift using an electron microscope. He referred to them as and as interchromatin particles. A connection at this point was not established. Swift observed that these particles were not randomly distributed, but appeared in localized "clouds". Cytochemical analyzes showed that they contained RNA. The first connection between pre-mRNA splicing and nuclear speckles or the interchromatin granule clusters emerged from an investigation of the distribution of snRNPs with anti-splicing factor-specific antibodies, which showed a speckled distribution pattern of snRNPs in cell nuclei. However, as research into the nuclear speckles advanced, additional functions were discovered.
construction
The human interphase nucleus contains 20–50 nuclear speckles with a diameter of one to several micrometers. By electron microscopic photographs, it was found that a single speckle of Nuclear points ( Interchromatin-Granule ) consists of 20-25 nm in diameter. These are connected to form a cluster by fine fibrils. Their size and shape can change dynamically. It can vary between different cell types and depends on many factors, including: a. cellular ATP levels, the phosphorylation status of various proteins, the transcription of stress-activated genes, and the transcription and splicing of RNA polymerase II. The inhibition of transcription by RNA polymerase II or splicing leads to the accumulation of proteins in enlarged nuclei Speckles. Once this inhibition has been removed, the normal size of the speckles can be restored. A target signal has been identified for some of the components of the speckles. The arginine / serine-rich domain (RS domain) of some SR pre-mRNA splicing factors has been shown to be necessary and sufficient to define speckles as targets for these proteins.
The biophysical properties of the nuclear speckles and the nucleoplasm do not differ significantly. Speckles are slightly denser than the surrounding nucleoplasm, and protein concentrations (between 115 and 162 mg / ml) are similar in both compartments. Although nuclear speckles are highly dynamic structures and their components are constantly in flux, speckles remain clearly separated from the nucleoplasm. When isolated from the nuclei of mouse liver cells, they remained stable and resistant to subsequent steps in the purification process.
It is generally accepted that the assembly of Nuclear Speckles depends on the interactions between their individual components. In addition to structured protein domains , low-complexity regions (LCRs) play an important role in protein-protein and protein-RNA interactions. LCRs are repetitive sequences of amino acids in proteins and are very flexible. These LCRs are over-represented in the proteins of the nuclear speckles. LCRs can permanently or temporarily change the properties of a protein after post-translational modification or in the event of protein partner binding . Proteins that contain LCRs are regulated and thus ensure that cellular processes can be adapted. The degradation or mutation in these LCR regions changes the protein-protein interactions, protein-RNA interactions, protein functions or the localization of proteins to the nuclear speckles.
The separation of proteins in the nuclear speckles enables them to protect them from unfavorable interactions and guarantees the integrity of the nuclear speckles despite the lack of a lipid boundary.
composition
Similar to other membraneless bodies with fluid-like properties, nuclear speckles are characterized by the dynamic exchange of components within the nucleoplasm. They share some proteins with other core bodies. For example, spliceosomal snRNPs are assembled in Cajal bodies before they are transported into the nuclear speckles . They also regulate the maturation of the 3 ′ ends of histone transcripts in histone locus bodies (HLB).
The number of proteins found in nuclear speckles has increased significantly through microscopic and proteomic studies. Nuclear speckle proteins are involved in several steps in nuclear gene expression regulation, such as: B. epigenetic regulation, transcription activator and repressor functions , transcription elongation and termination, splicing, 3 'end processing, mRNA modification and export. The localization, interactions and the breakdown of these proteins are regulated by regulatory proteins of the nuclear speckles. These include protein kinases and proteins involved in phosphoinositide signaling, cytoskeletal organization, and ubiquitination .
Protein kinases
Post-translational protein modifications that can alter protein properties are one of the most important functions of nuclear speckles. Based on their protein composition, Nuclear Speckles appear to be core centers for protein phosphorylation, methylation, acetylation, ubiquitination, and SUMOylation . Since more than 30% of human proteins can be phosphorylated and kinase-coding genes make up approximately 2.4% of protein-coding human genes, protein phosphorylation is a fundamental regulatory mechanism.
A total of 31 protein kinases were found in nuclear speckles (up to 2017) and the substrates of many protein kinases were identified. Since many proteins of the nuclear speckles are involved in different gene expression steps that take place in different cellular compartments, the control of the transfer of these proteins between the nucleus and cytoplasm has established itself as an important instrument of gene expression regulation. Reversible protein phosphorylation plays an important role in the correct cellular localization of nuclear speckle proteins, including SRSF proteins ( serine and arginine rich splicing factors ). SRSF phosphorylation / dephosphorylation serves as a critical mechanism affecting both splicing and protein composition in nuclear speckles. Accordingly, increased activity or overexpression of many nuclear speckle kinases leads to a breakdown of the speckles. This suggests a role for the kinases in the maintenance of the nuclear speckles. The kinases of the nuclear speckles are mediators that integrate and adapt various signals about nuclear gene expression regulatory events in order to maintain cellular equilibrium.
PI signaling
Phosphoinositol (PI) derivatives act as another large group of signaling molecules that are relevant to the function of nuclear speckles. In the nucleus, PIs are involved in the regulation of gene expression, including chromatin modification, pre-mRNA maturation, and mRNP export. Numerous nuclear speckle proteins are directly (with PI binding) or indirectly affected by PIs through the activity of PI-dependent regulatory proteins (e.g. protein kinases or ubiquitin ligases).
More and more evidence suggests that PIs are located in nuclear speckles. In addition, PI-modifying enzymes that add or remove phosphates from PIs, or hydrolyze them, interact with each other in nuclear speckles. This suggests that some PI derivatives are made in nuclear speckles. So far (2017) more than twenty proteins involved in PI signaling have been found in nuclear speckles.
PIs can have a significant impact on nuclear speckles function due to the functionality of their downstream signaling proteins, which include several prominent NS protein kinases. Taken together, these facts reinforce the view that nuclear speckles are nuclear hubs for PI production and signaling, important for protein localization in nuclear speckles and their components, and for the regulation of transcription, transcript maturation, splicing, and export.
Organization of the cytoskeleton
Various types of structural proteins have been identified in nuclear speckles in proteomic studies. Some studies have shown that cytoskeletal proteins such as lamines , myosins, and tubulin are components of nuclear speckles. Cytoskeletal rearrangements are regulated by nuclear speckles proteins, which are involved in PI signaling and calcium signaling. This suggests that the functional relationships between PIs, calcium and cytoskeletal proteins in the cytoplasm are similar in nuclear speckles. Proteins, which are involved in the organization of the cytoskeleton, contribute to the formation of the cell nucleus, but also regulate transcription.
Ubiquitination and SUMOylation
A significant proportion of the proteins in Nuclear Speckles are covalently bound to ubiquitin or ubiquitin-like proteins. Although active proteasomes play a role in protein breakdown in nuclear speckles and affect the assembly of nuclear speckles, only one type of polyubiquitination marks a protein for proteasomal breakdown. Ubiquitin-dependent regulation is an important mechanism for controlling splicing. Ubiquitination facilitates protein-protein interactions that are necessary for spliceosome formation.
In contrast, the SUMOylation of proteins from Nuclear Speckles is better understood. The binding of SUMO-1 is a typical signal used to bind proteins to a nuclear speckle. SUMOylation is involved in the regulation of many other processes associated with nuclear speckles.
RNAs
Numerous studies have demonstrated the presence of RNAs, including polyadenylated RNAs and non-coding RNAs, in nuclear speckles. A significant part of the total nuclear polyadenylated RNA is located within the nuclear speckles. The accumulation of some transcripts in nuclear speckles depends mainly on the presence of introns, but transcripts without introns also locate there. Nuclear export appears to be the most prominent pathway that regulates mRNAs residing in nuclear speckles, as exhaustion of complex components of transcription export (TREX) leads to improved association of mRNAs with nuclear speckles. Core mRNA export depends on active transcription, complete polyadenylation, and splicing. Since most exons are co-transcriptionally spliced at the transcription site, nuclear speckles appear to be the site of transcript maturation required for nuclear export.
MALAT1 RNA
A long, non-coding RNA called MALAT1 (metastasis-associated lung carcinoma transcript 1) plays an important role in the behavior of nuclear speckles. MALAT1 influences alternative splicing by regulating phosphorylation and the resulting nuclear distribution of splicing factors. It also recruits machinery for gene activation and the movement of active chromatin to the nuclear speckles. Several types of SRSFs proteins are associated with MALAT1. They bind directly to their recognition site at the end of 5 ′ of MALAT1. In addition, MALAT1 binds other proteins. Its localization to the nuclear speckles depends on various other proteins. It is known that MALAT1 directly interacts with nuclear RNAs such as U1 snRNA. MALAT1 also interacts with chromatin on actively spliced genes near polyadenylation sites.
Functions
Recent studies have shown that proteins involved in chromosome localization, chromatin modification, transcription, splicing, 3 ′ end processing, mRNA modification, and messenger ribonucleoprotein (mRNP) export are synthesized in the nuclear speckles. This supports the hypothesis that nuclear speckles act as a 'hub' to coordinate all steps in the regulation of nuclear gene expression.
All these steps are coupled with the transcription by the RNA polymerase II . This takes place in the immediate vicinity of the speckles. Despite many studies aiming to functionally characterize the proteins of nuclear speckles, further research is required to determine the role of the speckles. This need for additional studies also applies to extensively studied processes such as B. splicing, because in addition to the conventional view that nuclear speckles are involved in the synthesis, modification, intermediate storage and recycling of splicing factors, several reports have also shown splicing activity within the speckles.
Kinases, cytoskeletal proteins, and enzymes of ubiquitin and PI metabolism are prominent regulators of nuclear gene expression with a known role in transcription and splice regulation. However, they represent only a small part of the NS proteome. The largest group of nuclear speckle proteins are involved in transcription and include transcription factors and chromatin remodeling factors.
The formation of nuclear speckles and their function are closely linked to active transcription. RNA polymerase II integrates transcript synthesis with the DNA template and the DNA-regulating proteins on the one hand and RNA maturation and export on the other. In particular, the mRNA 3 ′ final processing and polyadenylation, mRNA m 6 A methylation, implementation of the mRNA core export and the chromatin regulation are also directly linked to the splicing.
Accordingly, more than 30 nuclear speckle proteins play an important role in both transcription and splicing. These processes are not only functionally linked, but also through physical protein-protein interactions.
It is believed that splicing affects the rate of RNA synthesis by promoting the pausing of RNA polymerase II . Conversely, the slowing down of RNA polymerase II promotes the accumulation of RNA polymerase II in the intron regions that flank the alternative exons. This enables the recruitment of activators or repressors of alternative splicing. These can promote the incorporation or passage of exons. Pre-mRNA splicing and negative regulation of transcription elongation are coupled by proteins localized to the nuclear speckle.
Epigenetic mechanisms of gene expression regulation serve as another important link between transcription and splicing. Transcription and splicing are largely involved in the regulation of chromatin structure and function. Since many DNA regulatory proteins have been found in nuclear speckles, the role of splicing factors and nuclear speckles in DNA regulation is an important target for future research.
mRNP ripening and export
Proteins located in nuclear speckles not only participate in numerous aspects of mRNA synthesis and mRNP maturation and are essential for the harmonization of these core processes, but also influence cytoplasmic mRNA behavior.
The last step in mRNA synthesis in almost all eukaryotes is the addition of a poly (A) chain at the 3 'end of the mRNA ( polyadenylation ). The most important protein factors that regulate and catalyze endonucleolytic cleavage and the subsequent polyadenylation are located in the nuclear speckles. It is believed that up to 70% of human mRNA transcripts undergo alternative polyadenylation. Nuclear speckle-associated processes, including CTD phosphorylation, epigenetic regulation, N6 methylation of adenosine (m 6 A), and splicing, are involved in choosing the right site in alternative polyadenylation. The m 6 A mRNA modification has a strong influence on the orchestration of mRNP maturation. The presence of all important nuclear elements of the m 6 A modification system in nuclear speckles indicates a prominent role of the speckles in m 6 A regulation.
The mutual relationship between mRNP maturation and the export mechanism can be viewed as part of the mRNA quality assurance system. This system affects the nuclear retention of transcripts that contain splicing, defective introns that form partial spliceosomes. This allows the export of only fully processed mRNPs. However, the protein composition of mRNPs controlled by nuclear speckles can also influence cytoplasmic RNA breakdown. Nuclear speckles regulate mRNP formation and the associated export efficiency.
Cell cycle
Nuclear speckles are very stable during interphase . However, the self-organizing properties of their components must be specifically suppressed during cell division. The disruption of the nuclear envelope after the initiation of mitosis leads to a breakdown of the nuclear speckles and a distribution of their proteins in the cytoplasm.
In addition to diffuse patterns observable at this point in time, the proteins of the nuclear speckles in the cytoplasm form an increasing number of mitotic interchromatin granules (MIGs), which are observed from metaphase to telophase. The MIGs are structurally similar to the Nuclear Speckles. Since the pre-mRNA splicing factors were in an active state immediately after entering the nucleus, it was postulated that the MIGs are required for the modification, assembly and transport of pre-mRNA processing complexes to transcription sites in daughter nuclei.
After the nuclear envelope is reconstructed, most pre-mRNA splicing factors gradually shift from MIGs to nucleus within 10 minutes, but some of them (e.g. SRSF2) can remain in MIGs until the G1 phase.
During telophase, before the formation of new nuclear speckles, splicing factors temporarily accumulate (for 15-20 min) in daughter nuclei in the vicinity of active nucleolus organizer regions , the so-called NOR-associated patches (NAPs). The assembly of the splicing factors into NAPs and MIGs confirms their strong self-organizing properties. However, the splicing factors alone are not enough to form nuclear speckles. Therefore, additional factors are required to produce nuclear speckles. Since the establishment of transcription in the telophase precedes the formation of the nuclear speckles, it was assumed that the recruitment of splicing and processing factors to the new active transcript sites triggers the spatial accumulation of nuclear speckles proteins, followed by the nucleation to build the speckles.
The accumulation of proteins to form nuclear speckles can be explained by a self-organization model based on protein-protein and protein-RNA interactions. However, initiation of the disintegration of the speckles during prophase requires additional unknown factors. Cyclins appear to be potential cell cycle regulators for speckles, as cyclin L1 is the only immobile protein in interphase nuclear speckles to date. Overall, the mechanisms that orchestrate the cell cycle-dependent build-up and breakdown of nuclear speckles are still a mystery and require further investigation.
Individual evidence
- ↑ a b c D. L. Spector, AI Lamond: Nuclear Speckles . In: Cold Spring Harbor Perspectives in Biology . tape 3 , no. 2 , February 1, 2011, ISSN 1943-0264 , p. a000646 – a000646 , doi : 10.1101 / cshperspect.a000646 , PMID 20926517 , PMC 3039535 (free full text).
- ↑ a b c d e f g h i j k l m n o p q r s t u Lukasz Galganski, Martyna O. Urbanek, Wlodzimierz J. Krzyzosiak: Nuclear speckles: molecular organization, biological function and role in disease . In: Nucleic Acids Research . tape 45 , no. 18 , October 13, 2017, ISSN 0305-1048 , p. 10350-10368 , doi : 10.1093 / nar / gkx759 , PMID 28977640 , PMC 5737799 (free full text) - ( oup.com [accessed April 24, 2019]).
- ↑ Figure 5: A 'regulated-exchange' model accounts for the dynamics of nuclear speckles. In: Nature Reviews Molecular Cell Biology . ISSN 1471-0080 ( nature.com [accessed May 1, 2019]).