Paraspeckle
Paraspeckles are irregularly shaped, membrane-less bodies in cell nuclei . They have a diameter of approx. 0.2–1 µm. There can be around 2–20 paraspeckles per nucleus, depending on the cell type. Their name is derived from their distribution in the core; "para" stands for parallel and "speckle" refers to the nuclear speckles that can always be found in close proximity.
Paraspeckles are critical to controlling gene expression by storing proteins and RNA . By acting as a "molecular sponge" mechanism, paraspeckles and their components can play a role in controlling gene expression during many cellular processes such as differentiation, viral infection and stress responses.
They are formed on the basis of a long, non-coding RNA called NEAT1. A number of studies have characterized their molecular components, internal structures and their cellular and physiological functions. Evidence suggests that the formation of paraspeckles is caused by phase separation of RNA-binding proteins. These contain disordered regions through which an ordered arrangement of paraspeckle components is mediated along the NEAT1 RNA.
Paraspeckles do not exist in human embryonic stem cells but only form after they have differentiated. Mice unable to produce NEAT1 RNA are viable under laboratory growth conditions, so paraspeckles are considered non-essential nuclear bodies. They are formed with certain environmental triggers such as viral infection, proteasome inhibition and differentiation. Paraspeckles are restricted to mammalian cell nuclei.
discovery
Paraspeckles were discovered in 2002 through a study of proteins in human nuclear bodies ( nucleolus ). Proteomic analysis of purified human nucleoli using mass spectrometry found that approximately 30% were novel proteins. Further analyzes on one of these newly identified proteins showed that they were not enriched in nucleoli, as expected, but were distributed diffusely within the caryoplasm . They were found concentrated in about 5–20 focal points in the cell nucleus. These points were observed in the vicinity of the Nuclear Speckles and were therefore referred to as "Paras-Speckles". Paraspeckles can also be identified as electron-dense structures in electron microscopy.
The novel protein that was located in these structures was later named "Paraspeckle Protein 1" (PSPC1). They also contained another protein called "NONO". Both proteins are members of the DHBS (Drosophila brain human splicing) family of RNA-binding proteins. Two other paraspeckle proteins, SFPQ and CFIm68, were discovered in later studies.
When cells were treated with drugs that inhibit the transcription of RNA polymerase II (Pol II), PSPC1 accumulated in crescent-shaped structures (“ perinucleolar cap ”). This was also found in the telophase of the cell cycle, in which no transcription of RNA polymerase II takes place. Here the paraspeckles dissolve and their components accumulate in the nucleoli. There the Paraspeckles proteins arrange themselves in a crescent shape and form the perinucleolar cap.
Involved RNA and Proteins
In addition to NEAT-RNA, paraspeckles contain several RNA-binding proteins. Three of these are members of the Drosophila Behavior Human Splicing (DBHS) family - NONO (P54NRB), SFPQ (PSF), and PSPC1 (PSP1). These DBHS proteins seem to play a key role in the structural integrity of paraspeckles: switching off the frequently present proteins NONO or SFPQ in HeLa cells led to a loss of paraspeckles. The proteins NONO, PSPC1 and SFPQ share about 50% of their sequence.
Paraspeckles also contain around 40 other associated proteins. These proteins are involved in several RNA metabolic pathways such as RNA processing and RNA stability. They bind to both double and single stranded DNA and RNA. They contain two RNA binding motifs at their amino terminal end.
The PSPC1 protein is most commonly used as a marker for paraspeckles. In addition, the ability to bind RNA is also required for the localization of PSPC1 to paraspeckles. The ability of PSPC1 to heterodimerize with NONO is necessary in order for it to localize to paraspeckles and the perinuclear caps.
The protein NONO has been detected in numerous nuclear events, including: a. the transcription regulation , splicing , DNA development, viral RNA processing and cellular proliferation .
Another function relevant to Paraspeckles is the involvement of SFPQ and NONO proteins in the nuclear retention of RNA molecules. This prevents an RNA processed by A-to-I editing from leaving the nucleus. In this mechanism, up to half of all adenosines (A) in the RNA are converted into inosines (I). This mostly occurs with double-stranded RNA molecules that contain inverted repeats in their nucleotide sequence. With what is believed to have emerged as an antiviral mechanism, the resulting inosine-containing RNA can be held in the nucleus instead of being exported to the cytoplasm .
Because of their role in processes such as splicing and transcription, the biological effects of DBHS proteins are far-reaching. Another example shows a conserved role of NONO in mammals in controlling the circadian rhythm. NONO is required for the maintenance of the circadian rhythm in mammals through association with the PERIOD-1 protein. DBHS proteins likely fulfill their diverse functions by varying their binding partners, performing post-translational modifications, and subcellular and subnuclear localizations.
Paraspeckle RNA
After the first discovery of paraspeckles, several observations suggested that paraspeckles contain RNA in addition to proteins. Two types of RNAs were identified that are specific for paraspeckles and each provide clues about their formation and function.
Ctn: role of paraspeckles in nuclear retention of RNA
The discovery of the first paraspeckle RNA in the mouse showed how these RNAs are involved in controlling gene expression by retaining the RNA in the cell nucleus. The mouse Ctn RNA is an alternative transcript that is generated from the mCAT2 gene. It's 8 kb long and is part of an antiviral response. In addition to its homogeneous distribution in the caryoplasm, it also localizes to paraspeckles.
Ctn differs from the mCAT2 mRNA in that it uses a different promoter and contains a much longer untranslated region (UTR) at its 3 'end . Both RNA variants behave in opposite ways: mCAT2 is exported from the core and translated normally. However, Ctn remains in the nucleus and paraspeckles of some cell types. The key to retaining this RNA in the paraspeckles lies in the long 3 ′ UTR region of Ctn. This contains double-stranded RNA hairpin structures that were changed by the A-to-I editing. The long 3 ′ UTR is only split off from Ctn by reacting to stress signals. Then the Ctn-RNA is exported to the cytoplasm, where it is translated by the ribosomes .
The cleavage event is associated with a simultaneous increase in the shorter mCAT2 mRNA in the cytoplasm and an increased protein production. Since the mCAT2 protein mediates the uptake of precursors in the NO reaction pathway, this release mechanism enables the cell to quickly activate an NO reaction to a stressful situation.
Although Ctn is absent in humans, this provides evidence that nuclear retention of RNA is widespread, as up to half of human transcripts could have expanded UTRs. A bioinformatic study has shown that many hundreds of human transcripts with inverted repeats are also available in a shorter form in which the inverted repeats have been removed. This suggests that the removal of inverted repeats in general can be used as a mechanism for releasing transcripts from nuclear storage.
NEAT1: a long non-coding RNA in paraspeckles
A long non-coding RNA ( lncRNA ) called NEAT1 acts as a scaffold for paraspeckles by recruiting many RNA-binding proteins that are involved in development, cancer, and neurodegeneration. NEAT1 is essential for the formation and maintenance of paraspeckles: in the absence of NEAT1 they do not reform again after transcription inhibition, and switching off the NEAT1 gene leads to the loss of paraspeckles.
Genes coding for NEAT1 and MALAT1 are typically located close together in mammalian genomes, somewhat removed from the nearest protein-coding gene. They are both ncRNA (non-coding RNA) accumulated in the cell nucleus . Another feature of NEAT1 and MALAT1 is that the long RNAs transcribed from each gene are both cleaved at their 3 'ends to produce an unusual small tRNA -like molecule. This could be a trademark of some nuclear ncRNAs. Both of these ncRNAs have been shown to localize to specific locations in the cell nucleus, MALAT1 within nuclear speckles and NEAT1 at locations that are adjacent to adjacent nuclear speckles. With NEAT1 it has been shown that they localize in paraspeckles and that the NEAT1 RNA is also essential for the integrity of the paraspeckles.
Two isoforms of NEAT1 are transcribed, NEAT1_v1 and NEAT1_v2 (also known as MEN-ϵ and MEN-β). They overlap in about 3-4 kb of their sequence on the 5 'side. Both transcripts localize in paraspeckles in mouse and human cells. The limitation of NEAT1 to paraspeckles is greater than that of DBHS proteins such as NONO, which is also abundant in the caryoplasm. NEAT1_v2 is stabilized by special triple helix structures. NEAT1_v2 forms the paraspeckle core, while NEAT1_v1 is used as a minor factor. While other protein components of paraspeckles are also found diffusely throughout the caryoplasm, the NEAT1-RNA occurs almost exclusively in paraspeckles.
NEAT1 probably also serves as a nucleation factor for paraspeckles. Paraspeckles were observed to form near the NEAT1 gene in the early G1 phase of the cell cycle. They are often clustered near the NEAT1 gene in the interphase. Like Ctn, NEAT1 associates with DBHS proteins. As with Ctn, it appears that interaction with DBHS proteins plays a role in NEAT1 paraspeckle localization. In contrast to Ctn, NEAT1 shows no evidence of A-to-I editing. This suggests that the DBHS proteins use more than one mode of RNA binding within paraspeckles. The presence of very sporadic short maintenance regions between mammalian NEAT1 sequences increases the possibility that DBHS proteins will bind to an RNA structure rather than a sequence. However, a previous study of the binding of DBHS proteins to U5SnRNA supports both sequence- and structure-based aspects of binding.
Structure and structure
Paraspeckles are small, irregular, and unevenly distributed subnuclear bodies. EM studies and fluorescence images show a paraspeckle size range of 0.5-1 µm in diameter and an irregular, sausage-like shape. So far, paraspeckles can only be found in mammalian cells. Paraspeckles are widespread within mammalian tissues and cells: the majority of mouse and human cell lines and tissues studied contain paraspeckles. They are observed in the interchromatin space and are virtually wedged between the larger nuclear speckles and chromatin. The functional relationship between paraspeckles and the nucleolus is not yet fully understood. In situ electron microscopy analyzes revealed that 5 ′ and 3 ′ end regions of NEAT1_2 are in the edge region of paraspeckles, while central regions of NEAT1_2 are in the core.
Paraspeckles consist of several components that can be divided into three groups: core, shell and patch components. The shell consists of regions 5 ′ and 3 ′ of NEAT1_2, Tardbp molecules (TDP-43) and purine-rich RNAs. NEAT1_1, the shorter isoform of NEAT1, is also in the shell. In the shell, regions 5 'and 3' of NEAT_2 do not mix and are observed as discrete points, suggesting that they are independently bundled. The core region of the paraspeckles consists of the RNA-binding proteins of the DBHS family (Sfpq, NONO and PSPC1) and Fus, as well as the middle region of NEAT1_2 RNAs. The patch components include Rbm14 and Brg1 proteins. They form several smaller patches that are distributed both in the shell and in the core of the paraspeckles. The subdivision of the paraspeckles into a core-shell structure could help that the core components in the caryoplasm are separated from the other components and are thus functionally inactivated by the sequestration. Additionally, the core components can play an active role in maintaining the structural integrity of the paraspeckle. Components of the shell could also offer a platform for certain molecular processes that take place on the periphery of paraspeckles
education
After the formation of paraspeckles, these remain largely unchanged during the cell cycle and only disappear when daughter nuclei form in the telophase. Research shows that the paraspeckles persist during the interphase and the time of mitosis, including prophase, metaphase, and anaphase. The formation of paraspeckles is initiated after cell division. Once paraspeckles have formed, they remain stable throughout the cell cycle and persist in mitosis through to anaphase when they spread across the cell. The formation of individual paraspeckles begins in the early G1 phase up to approx. 1 hour after the start of RNA transcription.
The formation of the paraspeckles could take place as follows: After cell division, the production of NEAT1 begins in the daughter nuclei. Before NEAT1 has the chance to break away from its locus, it is quickly picked up by DBHS protein dimers. Together, this RNA-protein complex builds up the paraspeckle particle. The finished paraspeckle will likely consist of multiple copies of NEAT1 RNA-DBHS protein complexes. These form a structural framework that is nevertheless dynamic: Individual DBHS protein molecules can be exchanged via the caryoplasm.
Without the production of NEAT1 RNA, no paraspeckles will form. This explains why paraspeckles are not observed when Pol II transcription is inhibited, either in cell types that do not express NEAT1 or in organisms that do not contain the NEAT1 gene. Conversely, without abundant DBHS proteins, no paraspeckles are observed.
The first paraspeckles form very close to the NEAT1 gene locus, and the paraspeckles also remain closely linked to the NEAT1 gene in the interphase; however, it is not known how the association is preserved.
Steps :
a) Paraspeckle formation begins with the production of NEAT1 transcripts in daughter nuclei after cell division.
b) After transcription, NEAT1 molecules form complexes with DBHS proteins, generally before the RNA has had the opportunity to diffuse far from its gene location. At some point, mutual interactions between these elements cause a phase transition along the basic units. This leads to the formation of spherical paraspeckles with radially arranged V-shaped NEAT1 base units. The phase transition should not be triggered by a simple increase in the concentration of paraspeckle proteins, but rather by a specific interaction and / or spatial arrangement of each component, which can be the basis for the formation of the ordered core-shell structure with a different diameter.
c) The finished paraspeckle probably consists of several copies of NEAT1 RNA-DBHS protein complexes that form a structural framework that is nevertheless dynamic in that individual DBHS protein molecules in paraspeckles can be exchanged with a pool of DBHS proteins in the caryoplasm.
function
A 2009 study (LLChen, GGCarmichael) combines the contributions of the three main types of protein and RNA molecules within paraspeckles in an important area of cell biology: pluripotency and differentiation. In this study, the authors conclude that the formation of paraspeckles is associated with a loss of pluripotency in embryonic stem cells. In addition, they suggest that a deficiency in NEAT1 and paraspeckles can be used as markers for pluripotency. Paraspeckles most likely contribute to differentiation by altering the gene expression profile through nuclear retention of A-to-I-edited mRNA.
At the molecular level, it has been found that paraspeckles can sequester proteins and RNA to modulate their behavior outside of the paraspeckles and thus act as a molecular sponge.They appear to be actively providing a platform for the assembly of molecular components necessary for certain molecular processes . The RNA retention mechanism can be involved in many cellular processes such as stress responses, viral infections, and the maintenance of the circadian rhythm.
Perhaps most importantly, given the link between paraspeckles and different models of differentiation, it is likely that paraspeckles play a role in reprogramming a cell that occurs with differentiation, possibly by altering the expression of key proteins via the storage of RNA in the nucleus.
Another possible role for paraspeckle in cell biology is the response to certain viruses. A previous study reported that VINC-1 (this has been shown to correspond to NEAT1) is upregulated in the central nervous system of mice after infection with Japanese encephalitis or rabies virus . Several studies have shown a connection between the upregulation of NEAT1 production and paraspeckle formation. So it is possible that these viruses can cause an increase in the size and number of paraspeckles, e.g. B. as a cellular defense mechanism.
Individual evidence
- ↑ a b c d e M. Dundr, T. Misteli: Biogenesis of Nuclear Bodies . In: Cold Spring Harbor Perspectives in Biology . tape 2 , no. 12 , December 1, 2010, ISSN 1943-0264 , p. a000711 – a000711 , doi : 10.1101 / cshperspect.a000711 , PMID 21068152 , PMC 2982170 (free full text).
- ↑ a b c d e f g h i j k l m n o p q r s t A. H. Fox, AI Lamond: Paraspeckles . In: Cold Spring Harbor Perspectives in Biology . tape 2 , no. 7 , July 1, 2010, ISSN 1943-0264 , p. a000687 – a000687 , doi : 10.1101 / cshperspect.a000687 , PMID 20573717 , PMC 2890200 (free full text).
- ↑ a b c d e f Kannanganattu V. Prasanth, Supriya G. Prasanth, Michael F. Jantsch, Tetsuro Hirose, Shinichi Nakagawa: Paraspeckles modulate the intranuclear distribution of paraspeckle-associated Ctn RNA . In: Scientific Reports . tape 6 , September 26, 2016, ISSN 2045-2322 , p. 34043 , doi : 10.1038 / srep34043 , PMID 27665741 , PMC 5036046 (free full text).
- ↑ a b c d e f g h i j k l Shinichi Nakagawa, Tomohiro Yamazaki, Tetsuro Hirose: Molecular dissection of nuclear paraspeckles: towards understanding the emerging world of the RNP milieu . In: Open Biology . tape 8 , no. October 10 , 2018, ISSN 2046-2441 , p. 180150 , doi : 10.1098 / rsob.180150 , PMID 30355755 , PMC 6223218 (free full text).
- ↑ a b c d e f g h i j k l m n o p q r s t u v w x y Charles S. Bond, Archa H. Fox: Paraspeckles: nuclear bodies built on long noncoding RNA . In: The Journal of Cell Biology . tape 186 , no. 5 , September 7, 2009, ISSN 0021-9525 , p. 637–644 , doi : 10.1083 / jcb.200906113 ( online [accessed June 8, 2019]).
- ↑ Jasmin Speil: Intracellular dynamics of the transcription factor STAT1. (PDF) July 22, 2010, accessed May 6, 2019 .
- ↑ a b c d e f Archa H. Fox, Charles S. Bond, Angus I. Lamond: P54nrb Forms a Heterodimer with PSP1 That Localizes to Paraspeckles in an RNA-dependent Manner . In: Molecular Biology of the Cell . tape 16 , no. November 11 , 2005, ISSN 1059-1524 , pp. 5304–5315 , doi : 10.1091 / mbc.e05-06-0587 , PMID 16148043 , PMC 1266428 (free full text).
- ↑ NONO non-POU domain containing octamer binding [Homo sapiens (human)]. Retrieved June 8, 2019 .
- ↑ SFPQ splicing factor proline and glutamine rich [Homo sapiens (human)]. Retrieved June 8, 2019 .
- ↑ PSPC1 paraspeckle component 1 [Homo sapiens (human)]. Retrieved June 8, 2019 .
- ↑ Miha Modic, Markus Grosch, Gregor Rot, Silvia Schirge, Tjasa Lepko: Cross-Regulation between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition . In: Molecular Cell . tape 74 , no. 5 , June 2019, p. 951–965.e13 , doi : 10.1016 / j.molcel.2019.03.041 ( online [accessed June 8, 2019]).
- ↑ Shinichi Nakagawa, Tetsuro Hirose: Paraspeckle nuclear bodies — useful uselessness? In: Cellular and Molecular Life Sciences . tape 69 , no. September 18 , 2012, ISSN 1420-682X , p. 3027–3036 , doi : 10.1007 / s00018-012-0973-x ( online [accessed June 8, 2019]).
- ↑ Ling-Ling Chen, Gordon G. Carmichael: Altered Nuclear Retention of mRNAs Containing Inverted Repeats in Human Embryonic Stem Cells: Functional Role of a Nuclear Noncoding RNA . In: Molecular Cell . tape 35 , no. 4 , August 2009, p. 467–478 , doi : 10.1016 / j.molcel.2009.06.027 ( online [accessed June 8, 2019]).