Ultrastructural localization of RNase A

and RNase L in cell models with different

transcriptional activity

by Sara Cortesi

Laboratory of Cell Biology and Neurobiology

University of Pavia

The nucleus

The nucleus of animal cells can be divided, from an ultrastructural point of view, into three different domains:

• Chromatin;

• Nucleolus;

• Nucleoplasm.

, 1994Fakan , 1969MonneronandBenhard

, , PF PG IG

Perichromatin f ibri ls ( ; + ), PF → which are the in situ form of nascent transcripts at the edge of condensed

chromatin

Perichromatin granules ( ; ), PG → produced by

.folding of hnRNPcontaining perichromatin fibrils

, 1969MonneronandBenhard

Interchromatin granules ( ), IG storage site of

, inactive factors involved in transcription for

. example splicing factors

Balance in transcription

95% .Approximately of hnRNAwill never undergo translation

.mRNA turnover performsan important role in regulation of geneexpression

., 2000Jacksonet al

, .ThesehnRNAsaredegraded in the nucleus even if theywere correctlymature

Ribonucleases

RNase A , 1938, discovered in can digest

.internucleotide linkages inRNAmolecules

RNase L is specifically induced in apoptosis and

- . activated by an interferon dipendent pathway

., 2006Ribóet al

., 2004Tanakaet al

ResearchHypothesis andApproach

The research hypothesis was that changes in the nuclear localization and quantities of

RNases A and L provide important information concerning their roles in the regulation of

. transcription

( ) Here I applied immunocytochemical immunogold labelling of thin sections and in situ

, hybridization techniques in cell and tissue models with different transcriptional activity both

. physiologically and as experimentally induced Statistical significance was determined by

- < 0.05. two wayANOVA andsignficancewas set at P

Primary antibodies Secondary antibodies Colloidal gold

a-RNase A (rabbit; 1:200) Goat-Anti-Rabbit (1:20) 12 nm

a-RNase L (mouse; 1:100) Goat-Anti-Mouse (1:20) 6 nm

a-hybrid (rabbit; 1:400) Goat-Anti-Rabbit (1:20) 12 nm

2 , (I also investigated whether peptides - ( )DADL Enkephalin DADLE and -DAL

( )Enkephalin DALE ), , known to downregulate transcription modify RNase A levels by

. immunogold labelling in HeLa cells DADLE treatment elicited decreased amounts of RNase A

48 . compared with controls and this effect was mantained after h of recovery DALE also evoked

.decreasedRNaseAbut by contrast thiswasnotmantainedafter recovery

RNaseA

HeLaDADLE + HeLaDADLE REC

Immunogold colocalizationRNaseA- (12 ) a RNaseA nm - (6 )a RNaseL nm

I also measured RNAse A content in thin

sections of thymus as a model of a tissue

. in which RNAse L becomes induced

RNase A quantity decreases in apoptotic

- thymocytes in comparison with non

.apoptotic thymocytes

: RNaseA regulation in thymus

RNase A levels increased in HeLa cells treated with heat shock (42 , 1 ) degC h or 5,6--1- - - (dichloro β D ribofuranosylbenzimidazole DRB ; a chemical blocker of the termination

) . of transcription if comparedwith control cells

: RNaseA regulation inHeLa cells

( ) ( )HeLa control HeLa Heat shock

Heat shock increases nuclearRNaseA levels- a RNase A - a RNase L

Previous work from our laboratory had shown altered transcription in male mice fed with

- - ( ) . glyphosate resistant genetically modified GM soy RNaseA increased in spermatocytes and

2 ( +).spermatids ofmice fed for monthswithmodified soy containingdiet GM

Feeding ofGMsoymodifiesRNaseAcontent

in cells ofmouse testis

Sertoli Spermatocytes Spermatids

GM- GM+ GM- GM+ GM- GM+

2 months ++ ++ °° °°° ** ***

8 months ++ ++ °° °° ** **

2 ( ) months control natural soy 2 monthsGMsoy

- RNaseA levels in spermatids of soy fedmice- a RNase A - a RNase L

- RNaseL inSertoli cells of soy fedmice

Sertoli cells of mice fed with transgenic soy

( +) GM present an increased quantity in

RNase L if compared with mice fed with a

( -). diet containingnatural soy GM

Sertoli Spermatocytes Spermatids

GM- GM+ GM- GM+ GM- GM+

2mm ++ +++ °° °° ** **

8mm ++ +++ °° °°° ** ***

8 - - Spermatocytes and spermatids from month old mice fed with a transgenic soy containing

( +) GM diet showed an increase in RNase L amount compared to those consuming a natural

- ( -). soy containing diet GM

- RNaseL content in testicular cells of soy fed

mice

Apoptotic thymocyte

In apoptotic thymocytes RNaseL - significantly increased in quantity in comparisonwith non

. apoptotic cells

RNaseL content increased in apoptotic

thymocytes

- Altered transcriptional activity in post hepatectomy liver

(1) RNase A amount varies dependently on cell t ranscript ional activity and .intensity of st imulus

, ( , ) Low intensity long lasting stimuli HeLaDADLE HeLaDALEand thymocytes

.causedecrease in enzymeamount

( , , 2 High intensity stimuli HeLa heat shock HeLaDRB hepatocytes and germ line cells of months

) .oldmice fedwithGMsoy induce increase inRNaseAquantity

(2) RNase L quantity increases only in 8 apoptotic thymocytes and in months .old mice fed with GM soy

: Conclusion regulation of RNase levels incells

: Conclusion RNase localization

- On perichromatin fibrils RNases probably work in a co transcriptional degradative

. mechanism

, . RNases also bind interchromatin granules in which they could be stored They could leave

. , these sites to reach PF and then come back to IG RNases could also digest PF that are often

.visualisedat periphery of IG

In further work not presented here because of time constraints, I showed the following:

(1) RNase A and RNase L bind perichromatin f ibri ls and interchromatin. granules

IG

PFRNase

PG