Structural Biochemistry/Nucleic Acid/RNA/RNA modification/RNA Degradation/RNaseT2 Functionality/

Introduction

Ribonucleases are important enzymes that catalyze the degradation of RNA in a broad range of organisms. Ribonucleases, otherwise referred to as RNase, have many families, one of which, the RNase T2 family, play many important roles in human pathologies. However, these functions of RNases of the T2 family are independent of its catalytic nuclease ability which will be covered below.

T2 Family Ribonuclease Specific Properties

Like many other RNases, RNaseT2 is secreted to compartments that are membrane-bound, such as the vacuole or lysosome. The RNases in this family cleave single stranded RNA (ssRNA). These RNases are transferase-type RNases and they cleave ssRNA in a 2',3'-cyclic phosphate intermediate. The result of this cleavage is the production or mono or oligonucleotides with a 3' terminal phosphate group.

There are three main differences between the T2 family and the A and T1 families. The T2 RNases are found in all kingdoms of organisms, from protozoans to animals and even viruses. However, the other families are localized to one or two kingdoms each. Additionally, the optimal pH of the T2 family is in the acidic range of 4-5 pH versus either alkaline or weakly basic ranges of the other families. This acidic optimization is consistent with the aforementioned localization to the lysosome or vacuole. This could indicate the cleavage occurring in acidic compartments is a function of this RNase. The third main difference is that T2 RNases cleave at all four bases while the other families are specific for either pyrimidines or a specific base.

T2 RNase Structure and Catalysis

T2 RNases have a conserved alpha/beta core structure on which regions of substrate binding have been found. These regions bind to mono or dinucleotides and these sites are conserved for the family of RNases. The two sites, B1 and B2, can be distinguished by being either 5' or 3' from the scissile bond, respectively. These sites, when occupied by nucleotides, function to position the phosphate bond which the active site of the enzyme can cleave. This cleaving ability is dependent upon a number of histidine residues in blocks of conserved amino acids in the T2 family. This ability can be inactivated in vitro and in vivo, allowing for the determination of protein functions is dependent on this catalytic ability. The catalytic cleavage by these RNases work in two steps called transphosphorylation and hydrolysis. The histidines performing this function alternately act as an acid and base during each step.

Biological Roles for T2 RNases

Although T2 RNases have the canonical role of cleaving ssRNA, they have a bevy of other functionalities that are expressed in vivo. Furthermore, many of these functions are independent of the catalytic ability of the T2 RNases.

Phosphate/Nucleic Acid Scavenging

These RNases have been correlated with the scavenging of nucleic acids outside of the cell for nutrients during cell starvation. It has been known that ribonucleases are induced when plant cells are starved for phosphates and that the extracellular activity of T2 RNases is increased. Furthermore a phosphodiesterase with increased activity and concentration during the condition of phosphate starvation has been found. Additionally, plants can use nucleic acids for a source of phosphates, as seen in the survival of plants with the only source of phosphate being yeast RNA. Although not confirmed, it would appear that the action of RNase T2 would allow the usage of extracellular nucleic acids as a phosphate source in the event of low phosphate conditions.

Additionally, RNaseT2 would appear to function as a recycler of cytoplasmic RNA that are delivered to the lysosomes and vacuoles in the event of autophagy. The source of these RNAs would be RNA granules that aggregate with proteins. Selective autophagy involving ubiquitin and ubiquitin-like modification recognition would allow for the targeting of RNA. P-granules, made up on proteins and RNA in germlin cells, interact with autophagy proteins and is necessary for degradation. One hypothesis is that mRNA that interact with these protein aggregates get degraded by RNaseT2 in acidic compartments and are moved their in autophagic processes.

Modulating Immune Response through Clearance of RNA

Mutations in RNaseT2 have been found to be the main inducer in an autosomal recessive disorder that causes cystic leukoencephalopathy. Although the mechanism behind this is unknown, it is also known that the symptoms presented are indistinguishable from the symptoms of infants infected with congenital cytomegalovirus (CMV). Thus, it could be possible that this infection triggers a change that is similar to that of an inactivated RNaseT2. It is known that CMV inactivates RNaseL in order to escape the host immune system. One hypothesis is that the failure to degrade intracellular ssRNA may trigger immune responses that lead to developmental consequences. This area is still the subject of intense research.

Parasitic Modulation of Host Immune Response

RNaseT2 is also known to play roles on the immune system in a direct manner as seen by the RNaseT2 secreted by Schistosoma mansoni, a parasitic blood fluke that lays eggs in a mammalian host. The eggs that this parasite lay secrete an RNaseT2 called omega-1. It has been known that omega-1 provokes an immune response that aids in the egg being excreted and eventually transmitted to a molluscan host intermediate. It has also been shown that omega-1 primes dendritic cells for Th2 polzarization of CD4+ T cells during an infection. As such, it would appear that omega-1 is taken up by dendritic cell and hence altering their functionality by altering their cytoskeletal structure.

Cytotoxicity

RNaseT2 can be cytotoxic as well to both other cells and the cell that. Furthermore these roles can be both catalytic dependent and catalytic-independent.

Analysis of pestiviruses such as classical swine fever virus shows that the envelope glycoprotein E^rns has an RNaseT2 domain and is secreted by infected cells. This enzyme was cytotoxic to lymphocytes and not epithelial cells, a result that indicates that E^rns serves to reduce the host immune response. This was shown to be catalytically dependent by the inactivation of the enzyme resulting in decreased viral infection. However, amino acid substitutions that still left the catalytic function intact prevented reduced host immune system levels. Overall, this shows that there are both catalytic and noncatalytic mechanisms that dampen the immune response through RNaseT2.

RNaseT2 cytotoxicity can also be seen in S-RNases which are found in certain plant families and serve to prevent self-pollination, among other pollen rejection mechanisms. These S-RNases enter the pollen tubes of both incompatible and compatible pollen tubes and degrade the pollen RNA of incompatible ones, causing disruption of self-fertilization. It has been shown that heat-inactivated S-RNase caused greater inhibition of self-pollination than intact S-RNase, indicating a catalytic-independent functionality. However, the mechanism behind how compatible pollen is protected is not completely elucidated and is undergoing continuing research.

However, early reports suggest that the pollen protein S-Locus F-box (SLF) is required for incompatibility and that S-RNase interacts with SLF. It has been proposed that SLF can cause proteasomal degradation of T2 RNases. However, this has not been confirmed.

Tumor Suppression and Growth

RNASET2 has been shown to be a tumor suppressor, as expression of this gene is reduced in both ovarian tumors and cancer cells. However, this conclusion is controversial. Additionally, reintroduction of RNASET2 into cells causes inhibition of clonogenicity of ovarian cancer cells in vitro. Furthermore, doing this has shown to reduce the metastatic potential of those cells. This ability has been shown to be catalytic-independent, as mutation of the enzyme still possessed anti-carcinogenic effects. However, research into this ability is still ongoing, as mutations within the coding region of RNASET2 in cancerous cells have not been identified.

References

1. Luhtala, N., & Parker, R. (2010). T2 Family ribonucleases: ancient enzymes with diverse roles. Trends in biochemical sciences, 35(5), 253–9. doi:10.1016/j.tibs.2010.02.002
2. Rosenberg HF. RNase A ribonucleases and host defense: an evolving story. J Leukoc Biol. 2008;83:1079–1087.
3. Luu DT, et al. S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility. Nature. 2000;407:649–651.